整合素CD11b抑制树突状细胞抗原交叉提呈的机制研究
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摘要
免疫活化和免疫耐受极大地依赖于抗原提呈的程度与免疫细胞的功能状态,作为专职的抗原提呈细胞和适应性免疫应答反应的始动者,树突状细胞(dendritic cell,DC)可以摄取、加工处理外源性抗原并以MHC-II类分子抗原肽复合物的形式提呈给CD4~+ T细胞。除此之外,DCs也可以将抗原以MHC-I类分子提呈给CD8~+T细胞,即细胞毒性T淋巴细胞(cytotoxic lymphocyte, CTL),这种特殊的抗原提呈方式被称为抗原交叉提呈反应(cross-priming)。已知DCs在病毒入侵、细菌感染以及肿瘤浸润的免疫防御反应中均发挥着重要的作用,而在此过程中,DCs分泌的IL-12p70不仅能促进CTL免疫突触的形成,还可以延长CTL-DC的相互作用从而促进CTL的增殖和IFN-γ的产生。DC初始化CTL的抗原交叉提呈反应可以在多个层面被调控,比如活化的共刺激信号和减弱的促凋亡信号均能增强抗原交叉提呈反应,但是仍有很多调控抗原交叉提呈反应的正向和负向调节子亟待发现和进一步深入研究。
β2整合素(CD11/CD18)在免疫反应和炎症过程中均发挥了重要的作用。根据其不同的α亚基,可以将其分为4种功能不同的异二聚体,分别为:白细胞功能相关性抗原(leukocyte function-associated antigen 1, LFA-1; CD11a/CD18,αLβ2 integrin, ITAL antigen), Mac-1 (CD11b/CD18,αMβ2 integrin, ITAM antigen), p150,95 (CD11c/CD18,αXβ2 integrin, CR4, ITAX antigen)和αDβ2 (CD11d/CD18, ITAD antigen)。其中CD11b广泛表达于多种免疫细胞亚群,如DCs、单核细胞、巨噬细胞、粒细胞和自然杀伤细胞等,并参与了细胞活化、细胞趋化、细胞毒和吞噬等多种生物学过程。本实验室的前期研究表明CD11b可以分别通过负向调控NK细胞的TLR3信号通路和巨噬细胞的TLR4信号通路来维持免疫耐受并抑制炎性反应。CD11b同样高表达于DC表面,已有研究表明DC表面的CD11b可以抑制DC初始的CD4~+ T细胞活化并通过破坏Th17的分化促进外周耐受的形成。这些结果表明CD11b在调控T细胞适应性免疫应答中的作用至关重要。在CTL活化过程中,普遍为大家多接受的是,TLR4配体LPS和TLR9配体CpG-ODN可以增强DC初始的CTL抗原交叉提呈反应,然而有关CD11b在抗原交叉提呈反应中的作用为何目前尚不知晓。
作为DC表面重要的两个TLR受体,TLR4和TLR9均可以促进DC成熟及其炎性因子分泌,但它们却依赖于不同的信号转导途径。在DC中,LPS主要以MyD88(myeloid differentiation factor 88)依赖的方式促进炎性因子的产生,而通过TRIF(TIR domain-containing adaptor inducing IFN-β)接头分子诱导共刺激分子的上调,如CD40、CD80和CD86等。TLR9启动信号级联反应则是特异性的通过接头分子MyD88来活化NF-κB和MAPK信号通路导致DC的成熟和炎性因子的分泌。
miRNAs(MicroRNAs)是一种非编码的具有调节功能的RNA,它们可以通过抑制转录和诱导mRNA降解在转录后水平调控基因的表达,在免疫反应中发挥着重要的调节功能。在天然免疫、炎症反应和病毒感染等进程中,miR-146a发挥着重要的调控作用。TLR信号通路的活化和炎性刺激均可以导致miR-146a的上调,继而依赖一些已知的靶调节免疫反应,如IL1-R-associated kinase (IRAK) 1, IRAK2, TNFR-associated factor (TRAF) 6和Tata Binding Protein (TBP)等。已有研究表明,miR-146a的上调主要是依赖于NF-κB活化,但其具体精细的调控机制知之甚少,此外,一个单独的miRNA可以靶向多个mRNA发挥生物学功能,所以仍然有很多miR-146a的潜在的靶亟待发现并鉴定其功能。
在本研究中,我们发现与正常对照小鼠DC相比,CD11b缺陷小鼠DC在TLR9激动剂刺激下IL-12p70明显上调,同样该DC初始的CTL抗原交叉提呈反应也显著增加,表现为CTL进一步增殖和IFN-γ分泌增加,这就表明CD11b在该过程中发挥着一种负向调节功能。进一步探讨其机制发现CD11b参与了TLR9介导的miR-146a上调,DC中上调的miR-146a进一步靶向Notch1从而抑制了IL-12p70的产生,因此,我们的实验结果揭示了CD11b具有抗原交叉提呈反应调控作用的新功能并对于其作用机制有了初步的认识。
1. CD11b通过抑制TLR9激动剂触发的IL-12p70而减弱抗原交叉提呈反应为了探究CD11b对抗原交叉提呈反应的影响,我们首先制备了正常小鼠和CD11b缺陷小鼠的骨髓来源的树突状细胞(Bone marrow-derived dendritic cells, BMDC),分别用TLR4激动剂LPS和TLR9激动剂CpG-ODN刺激一天诱导其成熟而作为抗原提呈细胞,然后分选OT-I小鼠脾脏CD8~+T细胞作为反应细胞,负载OVA抗原后与maDCs (mature DCs)共培养,以此来模拟抗原交叉提呈反应。我们发现,与对照组相比,CD11b缺陷小鼠在CpG-ODN刺激后可以通过促进CTL的增殖以及IFN-γ的分泌来增强抗原交叉提呈反应,而LPS刺激情况下则无明显改变,表明CD11b抑制了TLR9激动剂而非TLR4激动剂诱导的抗原交叉提呈反应。
为了进一步明确其具体机制,我们首先利用流式细胞仪对比了正常小鼠和CD11b缺陷小鼠DC的表型,结果无论是imDCs (immature DCs)还是LPS或CpG-ODN刺激后的DC,其上的MHC-I、MHC-II、CD40、CD70、CD80和CD86均没有明显改变,表明CD11b并非通过影响DC表型发挥作用,随后借助ELISA,我们对比了两种DC在TLR4和TLR9激动剂刺激后IL-6、TNF-α和IL-1β的表达情况,结果依然没有显著差异,但是CD11b缺陷小鼠DC在TLR9激动剂刺激情况下IL-12p70却显著上调,且特异性IL-12p70中和性抗体能减弱正常小鼠DC和CD11b缺陷小鼠DC所诱导的抗原交叉提呈反应并缩小其组间差异,因为诸多研究表明IL-12p70可以增强CTL活化,所以,该部分结果表明CD11b通过抑制TLR9激动剂触发的IL-12p70进而减弱抗原交叉提呈反应。
2. CD11b促进CpG-ODN诱导的miR-146a上调依赖于NF-κB的晚期活化CD11b缺失后只改变了IL-12p70的分泌而对DC表型和炎性因子产生均无明显影响,表明CD11b很有可能并非直接调控DC内TLR9信号通路,而是借助一些其他途径和机制特异性的调节了IL-12p70的产生。
鉴于miRNA可以在转录后水平调控基因表达,我们挑选了一些候选miRNA进行检测来验证是否CD11b通过改变miRNA的表达调控了IL-12p70的分泌。据文献报道,miR-21可以直接调控IL-12p35,然而在我们的体系中CD11b缺陷与否对其表达丰度并无影响,miR-146a和miR-155都可以在TLR配体刺激下上调,通过实时定量PCR我们发现,无论CD11b缺陷与否,LPS和CpG-ODN均能有效的诱导miR-155上调,而DC中CD11b缺失后特异性的抑制了CpG-ODN诱导的miR-146a上调,而对LPS刺激的miR-146a上调无影响,且pri-miR-146a的表达趋势与miR-146a一致表明CD11b在转录水平参与调控了miR-146a的上调。此外NF-κB抑制剂PDTC可以有效的阻断正常小鼠DC中CpG-ODN诱导的miR-146a上调,提示CD11b以NF-κB依赖的方式辅助了miR-146a上调。同时通过免疫蛋白印迹实验我们动态观测了CpG-ODN刺激后,正常小鼠DC和CD11b缺陷小鼠DC的NF-κB活化情况,结果发现CD11b并未影响早期的NF-κB的活化,从而解释了炎性因子没有改变的原因,而CD11b缺失后NF-κB的晚期活化明显减弱,导致了miR-146a无法上调。
3. miR-146a通过靶向Notch1抑制CpG-ODN诱导DC产生的IL-12p70 miRNA主要通过靶向mRNA发挥生物调节功能,且已知的miR-146a的靶IRAK1、IRAK2、TRAF6和TBP貌似不会调控IL-12p70的分泌,所以很有可能存在miR-146a的新靶调控了IL-12p70的表达,通过TargetScan(http://www.targetscan.org),我们对miR-146a的作用靶分子进行了预测,并发现Notch1在各种属间拥有保守的miR-146a结合位点,且已有研究表明TLR的活化可以激活Notch1信号,且Notch1的活化与M1型巨噬细胞高分泌IL-12p70存在正相关,提示miR-146a很有可能靶向Notch1抑制IL-12p70的产生,通过报告基因实验对其进行进一步验证发现,与mimic control相比miR-146a mimic能显著抑制含miR-146a结合位点的Notch1的3’UTR区正常报告基因质粒的活性,而对结合位点突变组无明显差别,表明miR-146a确实可以靶向Notch1。
接下来我们比较了对照组与CD11b缺陷组DC的Notch1全长片段(Notch FL)和Notch1胞内段(Notch intracellular domain, NICD)的固有表达情况以及在CpG-ODN刺激后的表达情况,结果显示,静息情况下,CD11b缺陷组DC的Notch FL和NICD1的表达即高于对照组,而CpG-ODN刺激后Notch FL和NICD1均明显上调且在CD11b缺陷组上调更为明显,表明CD11b很有可能通过抑制Notch1的表达来减少IL-12p70的分泌。此外,为了验证miR-146a对Notch1的影响,在将miR-146a mimic、mimic control、miR-146a inhibitor和inhibitor control转染至CD11b缺陷DC和正常DC之后我们分别在RNA水平和蛋白水平检测了Notch1的表达情况,结果与预期的情况一致,miR-146a mimic明显抑制了Notch1的表达,而miR-146a inhibitor正好相反,表明在DC中Notch1确实是miR-146a的一个新靶,通过转录抑制和mRNA降解的机制被miR-146a所调控。
随后通过RNA干扰实验我们发现,无论是在正常小鼠DC还是CD11b缺陷小鼠DC,干扰Notch1后可明显降低CpG-ODN诱导的IL-12p70的分泌,表明Notch1参与了CpG-ODN诱导的IL-12p70产生。最后为了验证miR-146a是否对CpG-ODN诱导的IL-12p70同样具有调控作用,我们在转染miRNA之后检测了IL-12p70的变化,结果显示转染miR-146a mimic过表达miR-146a后,较正常组CD11b缺陷小鼠DC组CpG-ODN刺激分泌的IL-12p70显著下调,而较CD11b缺陷小鼠DC正常小鼠DC转染miR-146a inhibitor后IL-12p70明显上调,可见CD11b的确通过辅助miR-146a的上调继而靶向Notch1来抑制了CpG-ODN触发的IL-12p70产生。
结语:
综上所述,CD11b参与、辅助了CpG-ODN诱导DC中miR-146a上调并由此靶向Notch1继而抑制了IL-12p70的产生,最终减弱了DC的抗原交叉提呈反应。本研究提供了整合素调控抗原交叉提呈反应的直接证据,并发现了miR-146a的新的生物学靶及其新功能,该结果不仅对CD11b调控miR-146a的机制进行了探讨分析,更为今后临床抗肿瘤、抗感染治疗中如何提升DC疫苗活性来增强抗原交叉提呈反应提供了新的靶点。
Immune activation or immune tolerance largely depends on the degree of antigen presentation and functional state of the immune cells involved. As specialized antigen-presenting cells and initiator of adaptive immune response, dendritic cells (DCs) can take up, process, and present exogenous antigens with MHC class II molecules to CD4~+ T cells. Apart from this, DCs can also present exogenous antigens to CTLs via MHC class I molecules. This special antigen presentation, known as cross-priming, has been shown to play critical roles in the immune defense against virus, bacteria, and tumor. During this process, IL-12p70, predominantly produced by DCs, not only enhances CTL synapse formation and prolongs CTL-DC interactions, but also promotes CTL proliferation and IFN-γproduction. DC cross-priming of CTL response is mediated by multiple mechanisms, such as activated costimulatory signals and attenuated proapoptotic signals. However, identifying positive and negative regulators in this process is still an ongoing process.
β2 integrins (CD11/CD18) play important roles in immune response and inflammation. According to their distinctαsubunits,β2 integrins are divided into four functional heterodimers termed leukocyte function-associated antigen 1 (LFA-1; CD11a/CD18,αLβ2 integrin, ITAL antigen), Mac-1 (CD11b/CD18,αMβ2 integrin, ITAM antigen), p150,95 (CD11c/CD18,αXβ2 integrin, CR4, ITAX antigen), andαDβ2 (CD11d/CD18, ITAD antigen). CD11b is extensively expressed in most immune cells, such as DC, monocytes, macrophages, granulocytes, and NK cells, participating in cell activation, chemotaxis, cytotoxicity, and phagocytosis. Our previous studies have shown that CD11b is able to maintain immunological tolerance and inhibit inflammatory responses via repressing TLR3 signaling in NK cells and TLR4 signaling in macrophages. Analogously, CD11b, abundantly expressed in DCs, can inhibit DC-primed CD4~+ T cell activation and promote peripheral tolerance by breaking Th17 differentiation. These reports have suggested the roles of CD11b in the regulation of T cell adaptive immunity. However, it remains unclear whether CD11b is also involved in DC cross-priming for CTL response. In CTL activation, it is well-accepted that TLR ligands, including LPS and CpG-ODN, are able to improve vaccination aiming at induction of CTLs, but the role of CD11b in this process is still unknown.
Although TLR4 and TLR9 both contribute to DC maturation and cytokine production, they depend on different signaling pathways. In DCs, LPS provokes cytokine production mainly through myeloid differentiation factor 88 (MyD88) dependent manner while induces upregulation of costimulatory molecules including CD40, CD80, and CD86 via the TIR domain-containing adaptor inducing IFN-β(TRIF) dependent manner. But TLR9 exclusively initiates a signaling cascade through MyD88, which activates NF-κB and MAPK pathways, and in turn induces proinflammatory cytokine production and DC maturation.
Micro-RNAs (miRNAs) are a class of small non-coding regulatory RNAs. They control gene expression at the post-transcriptional level by inhibiting translation or inducing mRNA degradation. miRNAs are emerging as important regulators in immune responses. Among them, miR-146a plays key roles in innate immunity, inflammatory response, virus infection, and human diseases. Both TLR activation and inflammatory stimulation can lead to upregulation of miR-146a, which in turn negatively regulates innate immunity by repressing its targets, such as IL1-R-associated kinase (IRAK) 1, IRAK2, TNFR-associated factor (TRAF) 6, and Tata Binding Protein (TBP). The induction of miR-146a is reported to be dependent on NF-κB activation, but the precise regulatory mechanism remains unclear. Apart from this, as a single miRNA has been thought to target multiple mRNAs, there are still many potential targets of miR-146a remain to be identified.
Here, in CD11b-deficient DCs, we found that TLR9-triggered IL-12p70 production was increased, and DC cross-priming for CTL response was elevated, suggesting the negative regulation of CD11b in DC cross-priming. For the mechanisms responsible for its negative regulation, we showed that CD11b participates in TLR9-mediated miR-146a upregulation in DCs. And the upregulated miR-146a in turn targets Notch1 to repress IL-12p70 production. Hence, our results reveal a novel role of CD11b in DC cross-priming for CTL response.
1. CD11b represses DC cross-priming of CTL response by inhibiting IL-12p70 production
To investigate the involvement of CD11b in DC cross-priming CTL response, we cultured BMDCs from wild-type or CD11b-deficient mice, and stimulated with TLR4 ligand (LPS) or TLR9 ligand (CpG-ODN) for 24 hours. When loaded with antigen OVA and co-cultured with na?ve CD8+ CTLs from OT-I mice, CD11b-deficient BMDCs had significantly enhanced CpG-triggered DC cross-priming of CTL response compared with that of wild-type BMDCs, as shown by more OVA-specific CTLs and higher IFN-γproduction. Unexpectedly, CD11b-deficiency did not affect LPS-triggered DC cross-priming CTL response. These results indicate that CD11b in DCs can negatively regulate TLR9, but not TLR4, -triggered cross-priming CTL response.
To further clarify the specific mechanism, we firstly detected the phenotype of CD11b-deficient DCs upon LPS or CpG-ODN stimulation. FACS analysis showed that CD11b-deficiency had little effect on the expression of MHC-I, MHC-II, CD40, CD70, CD80, or CD86 on both immature and mature BMDCs, thus indicating that CD11b mediated negative regulation of DC cross-priming CTL response was independent of cell surface costimulatory molecules. After screening of cytokines production in LPS or CpG-ODN stimulated DCs, we found that CD11b-deficiency had no significant influence on IL-6, TNF-α, or IL-1βproduction. Interestingly, CpG-ODN-induced IL-12p70 production was significantly enhanced in CD11b-deficient BMDCs than that in wild-type BMDCs, furthermore, specific neutralizing antibody to IL-12p70 diminished the differed CTL proliferation and IFN-γproduction between CD11b-deficient and wild-type DCs upon CpG-ODN stimulation. Because IL-12p70 is well-accepted to augment CTL activation, these results demonstrate that CD11b negatively regulates CpG-ODN-induced IL-12p70 production in DCs, and in turn represses DC cross-priming CTL response.
2. CD11b participates in CpG-ODN-induced miR-146a upregulation in DCs via sustaining late-phase NF-κB activation
In CpG-ODN-triggered DC cross-priming CTL response, CD11b only negatively regulates CpG-ODN-induced IL-12p70 production, but does not influence DC phenotype maturation or inflammatory cytokines production. This phenomenon indicates that CD11b is less likely to directly influence TLR9 signaling in DCs. So, some other indirect mechanisms may be responsible for the enhanced IL-12p70 production in CD11b-deficient DCs upon CpG-ODN stimulation.
miRNAs are important regulators in immune response. Thus, we wondered whether CD11b is involved in the regulation of miRNAs expression induced by CpG-ODN in DCs, which may subsequently contribute to the decreased IL-12p70 production. To further verify this hypothesis, we selected some miRNAs as candidates, especially those identified to be important regulators in TLR response. In details, miR-21 is known to regulate IL-12p35 expression, but CD11b-deficiency does not seem to affect miR-21 expression in LPS or CpG-ODN-stimulated DCs; miR-155 is another TLR signaling induced miRNA, and CD11b-deficiency has no significant effect on its expression either; for miR-146a, known to be upregulated upon TLR agonists stimulation, CD11b-deficiency inhibited CpG-ODN-induced miR-146a upregulation. Apart from this, in wild-type DCs, both LPS and CpG-ODN induced pri-miR-146a expression, and CD11b-deficiency also abolished CpG-ODN, but not LPS, -triggered pri-miR-146a expression, which suggests that miR-146a is regulated by CD11b at the transcriptional level. Since induction of miR-146a has been proved to be NF-κB dependent, we also validated that CpG-ODN-induced miR-146a expression was dependent on NF-κB activation, shown as NF-κB inhibitor inhibited its expression upon CpG-ODN stimulation in DCs. Furthermore, we determined the NF-κB activation by immunoblot, we found that CD11b doesn’t influence CpG-ODN-induced early-phase NF-κB activation, thus has no effect on proinflammatory cytokine production; but participates in CpG-ODN-induced late-phase NF-κB activation, and in turn upregulates miR-146a expression in DCs.
3. miR-146a represses CpG-ODN-induced IL-12p70 production by targeting Notch1 in DCs
As miRNAs function mainly through repressing their target mRNAs, and the known targets of miR-146a, such as IRAK1, IRAK2, TRAF6, and TBP, seem to be unrelated to IL-12p70 production, we searched new possible targets of miR-146a through TargetScan prediction (http://www.targetscan.org). Among the putative targets of miR-146a, we found that Notch1 had a putative conserved miR-146a target site. Notch signaling has been shown to be induced by TLR activation and participate in TLR activation-induced IL-12p70 production. To certify whether Notch1 3’UTR was directly targeted by miR-146a, we constructed a reporter plasmid containing Notch1 3’UTR and found that its expression was inhibited by miR-146a co-transfection, while the seed region mutated construct failed to be inhibited by miR-146a expression. This result suggests that Nortch1 is directly targeted by miR-146a expression.
In addition, in CpG-ODN-stimulated DCs, expression of full length Notch1 and Notch intracellular domain (NICD) 1 were increased by CD11b-deficiency, suggesting that CD11b may repress IL-12p70 production via inhibiting Notch1. To verify the impact of miR-146a on Notch1, we transfected miRNA into DCs, and miR-146a mimic markedly reduced CpG-ODN-induced Notch1 expression at both protein and mRNA levels, whereas miR-146a inhibitor increased their expression. Taken together, these results suggest that Notch1, as a new target of miR-146a, may be responsible for the downregulated IL-12p70 production by CD11b expression.
Next we examined the effects of Notch1 knockdown on CpG-ODN-triggered IL-12p70 production. Notch1 specific siRNA inhibited the expression of both full length Notch1 and NICD1, and Notch1 knockdown weakened IL-12p70 production in both wild-type and CD11b-deficient DCs upon CpG-ODN stimulation. These data suggests that Notch1 participates in CpG-ODN-triggered IL-12p70 production. We next determined whether miR-146a was responsible for the upregulated IL-12p70 production in CD11b-deficient DCs upon CpG-ODN stimulation. Transfection of miR-146a inhibitor promoted CpG-ODN induced IL-12p70 production in wild-type DCs but did not influence that in CD11b-deficient DCs. These data further confirm that CD11b represses CpG-ODN-triggered IL-12p70 production via upregulating miR-146a expression and targeting Notch1 in DCs.
Conclusion
Taken together, we demonstrate that CD11b is a negative regulator of TLR9 triggered DC cross-priming of CTL response via inhibiting IL-12p70 production in DC, which is mediated by upregulating miR-146a and targeting Notch1. Our study presents a new model in the negative regulation of TLR9 agonist-triggered DC cross-priming of CTL response by CD11b. Hence, CD11b and downstream miR-146a may be new regulators in DC licensed cross-priming aimed at induction of CTLs for the treatment of infectious diseases and cancer.
β2整合素(CD11/CD18)在免疫反应和炎症过程中均发挥了重要的作用。根据其不同的α亚基,可以将其分为4种功能不同的异二聚体,分别为:白细胞功能相关性抗原(leukocyte function-associated antigen 1, LFA-1; CD11a/CD18,αLβ2 integrin, ITAL antigen), Mac-1 (CD11b/CD18,αMβ2 integrin, ITAM antigen), p150,95 (CD11c/CD18,αXβ2 integrin, CR4, ITAX antigen)和αDβ2 (CD11d/CD18, ITAD antigen)。其中CD11b广泛表达于多种免疫细胞亚群,如DCs、单核细胞、巨噬细胞、粒细胞和自然杀伤细胞等,并参与了细胞活化、细胞趋化、细胞毒和吞噬等多种生物学过程。本实验室的前期研究表明CD11b可以分别通过负向调控NK细胞的TLR3信号通路和巨噬细胞的TLR4信号通路来维持免疫耐受并抑制炎性反应。CD11b同样高表达于DC表面,已有研究表明DC表面的CD11b可以抑制DC初始的CD4~+ T细胞活化并通过破坏Th17的分化促进外周耐受的形成。这些结果表明CD11b在调控T细胞适应性免疫应答中的作用至关重要。在CTL活化过程中,普遍为大家多接受的是,TLR4配体LPS和TLR9配体CpG-ODN可以增强DC初始的CTL抗原交叉提呈反应,然而有关CD11b在抗原交叉提呈反应中的作用为何目前尚不知晓。
作为DC表面重要的两个TLR受体,TLR4和TLR9均可以促进DC成熟及其炎性因子分泌,但它们却依赖于不同的信号转导途径。在DC中,LPS主要以MyD88(myeloid differentiation factor 88)依赖的方式促进炎性因子的产生,而通过TRIF(TIR domain-containing adaptor inducing IFN-β)接头分子诱导共刺激分子的上调,如CD40、CD80和CD86等。TLR9启动信号级联反应则是特异性的通过接头分子MyD88来活化NF-κB和MAPK信号通路导致DC的成熟和炎性因子的分泌。
miRNAs(MicroRNAs)是一种非编码的具有调节功能的RNA,它们可以通过抑制转录和诱导mRNA降解在转录后水平调控基因的表达,在免疫反应中发挥着重要的调节功能。在天然免疫、炎症反应和病毒感染等进程中,miR-146a发挥着重要的调控作用。TLR信号通路的活化和炎性刺激均可以导致miR-146a的上调,继而依赖一些已知的靶调节免疫反应,如IL1-R-associated kinase (IRAK) 1, IRAK2, TNFR-associated factor (TRAF) 6和Tata Binding Protein (TBP)等。已有研究表明,miR-146a的上调主要是依赖于NF-κB活化,但其具体精细的调控机制知之甚少,此外,一个单独的miRNA可以靶向多个mRNA发挥生物学功能,所以仍然有很多miR-146a的潜在的靶亟待发现并鉴定其功能。
在本研究中,我们发现与正常对照小鼠DC相比,CD11b缺陷小鼠DC在TLR9激动剂刺激下IL-12p70明显上调,同样该DC初始的CTL抗原交叉提呈反应也显著增加,表现为CTL进一步增殖和IFN-γ分泌增加,这就表明CD11b在该过程中发挥着一种负向调节功能。进一步探讨其机制发现CD11b参与了TLR9介导的miR-146a上调,DC中上调的miR-146a进一步靶向Notch1从而抑制了IL-12p70的产生,因此,我们的实验结果揭示了CD11b具有抗原交叉提呈反应调控作用的新功能并对于其作用机制有了初步的认识。
1. CD11b通过抑制TLR9激动剂触发的IL-12p70而减弱抗原交叉提呈反应为了探究CD11b对抗原交叉提呈反应的影响,我们首先制备了正常小鼠和CD11b缺陷小鼠的骨髓来源的树突状细胞(Bone marrow-derived dendritic cells, BMDC),分别用TLR4激动剂LPS和TLR9激动剂CpG-ODN刺激一天诱导其成熟而作为抗原提呈细胞,然后分选OT-I小鼠脾脏CD8~+T细胞作为反应细胞,负载OVA抗原后与maDCs (mature DCs)共培养,以此来模拟抗原交叉提呈反应。我们发现,与对照组相比,CD11b缺陷小鼠在CpG-ODN刺激后可以通过促进CTL的增殖以及IFN-γ的分泌来增强抗原交叉提呈反应,而LPS刺激情况下则无明显改变,表明CD11b抑制了TLR9激动剂而非TLR4激动剂诱导的抗原交叉提呈反应。
为了进一步明确其具体机制,我们首先利用流式细胞仪对比了正常小鼠和CD11b缺陷小鼠DC的表型,结果无论是imDCs (immature DCs)还是LPS或CpG-ODN刺激后的DC,其上的MHC-I、MHC-II、CD40、CD70、CD80和CD86均没有明显改变,表明CD11b并非通过影响DC表型发挥作用,随后借助ELISA,我们对比了两种DC在TLR4和TLR9激动剂刺激后IL-6、TNF-α和IL-1β的表达情况,结果依然没有显著差异,但是CD11b缺陷小鼠DC在TLR9激动剂刺激情况下IL-12p70却显著上调,且特异性IL-12p70中和性抗体能减弱正常小鼠DC和CD11b缺陷小鼠DC所诱导的抗原交叉提呈反应并缩小其组间差异,因为诸多研究表明IL-12p70可以增强CTL活化,所以,该部分结果表明CD11b通过抑制TLR9激动剂触发的IL-12p70进而减弱抗原交叉提呈反应。
2. CD11b促进CpG-ODN诱导的miR-146a上调依赖于NF-κB的晚期活化CD11b缺失后只改变了IL-12p70的分泌而对DC表型和炎性因子产生均无明显影响,表明CD11b很有可能并非直接调控DC内TLR9信号通路,而是借助一些其他途径和机制特异性的调节了IL-12p70的产生。
鉴于miRNA可以在转录后水平调控基因表达,我们挑选了一些候选miRNA进行检测来验证是否CD11b通过改变miRNA的表达调控了IL-12p70的分泌。据文献报道,miR-21可以直接调控IL-12p35,然而在我们的体系中CD11b缺陷与否对其表达丰度并无影响,miR-146a和miR-155都可以在TLR配体刺激下上调,通过实时定量PCR我们发现,无论CD11b缺陷与否,LPS和CpG-ODN均能有效的诱导miR-155上调,而DC中CD11b缺失后特异性的抑制了CpG-ODN诱导的miR-146a上调,而对LPS刺激的miR-146a上调无影响,且pri-miR-146a的表达趋势与miR-146a一致表明CD11b在转录水平参与调控了miR-146a的上调。此外NF-κB抑制剂PDTC可以有效的阻断正常小鼠DC中CpG-ODN诱导的miR-146a上调,提示CD11b以NF-κB依赖的方式辅助了miR-146a上调。同时通过免疫蛋白印迹实验我们动态观测了CpG-ODN刺激后,正常小鼠DC和CD11b缺陷小鼠DC的NF-κB活化情况,结果发现CD11b并未影响早期的NF-κB的活化,从而解释了炎性因子没有改变的原因,而CD11b缺失后NF-κB的晚期活化明显减弱,导致了miR-146a无法上调。
3. miR-146a通过靶向Notch1抑制CpG-ODN诱导DC产生的IL-12p70 miRNA主要通过靶向mRNA发挥生物调节功能,且已知的miR-146a的靶IRAK1、IRAK2、TRAF6和TBP貌似不会调控IL-12p70的分泌,所以很有可能存在miR-146a的新靶调控了IL-12p70的表达,通过TargetScan(http://www.targetscan.org),我们对miR-146a的作用靶分子进行了预测,并发现Notch1在各种属间拥有保守的miR-146a结合位点,且已有研究表明TLR的活化可以激活Notch1信号,且Notch1的活化与M1型巨噬细胞高分泌IL-12p70存在正相关,提示miR-146a很有可能靶向Notch1抑制IL-12p70的产生,通过报告基因实验对其进行进一步验证发现,与mimic control相比miR-146a mimic能显著抑制含miR-146a结合位点的Notch1的3’UTR区正常报告基因质粒的活性,而对结合位点突变组无明显差别,表明miR-146a确实可以靶向Notch1。
接下来我们比较了对照组与CD11b缺陷组DC的Notch1全长片段(Notch FL)和Notch1胞内段(Notch intracellular domain, NICD)的固有表达情况以及在CpG-ODN刺激后的表达情况,结果显示,静息情况下,CD11b缺陷组DC的Notch FL和NICD1的表达即高于对照组,而CpG-ODN刺激后Notch FL和NICD1均明显上调且在CD11b缺陷组上调更为明显,表明CD11b很有可能通过抑制Notch1的表达来减少IL-12p70的分泌。此外,为了验证miR-146a对Notch1的影响,在将miR-146a mimic、mimic control、miR-146a inhibitor和inhibitor control转染至CD11b缺陷DC和正常DC之后我们分别在RNA水平和蛋白水平检测了Notch1的表达情况,结果与预期的情况一致,miR-146a mimic明显抑制了Notch1的表达,而miR-146a inhibitor正好相反,表明在DC中Notch1确实是miR-146a的一个新靶,通过转录抑制和mRNA降解的机制被miR-146a所调控。
随后通过RNA干扰实验我们发现,无论是在正常小鼠DC还是CD11b缺陷小鼠DC,干扰Notch1后可明显降低CpG-ODN诱导的IL-12p70的分泌,表明Notch1参与了CpG-ODN诱导的IL-12p70产生。最后为了验证miR-146a是否对CpG-ODN诱导的IL-12p70同样具有调控作用,我们在转染miRNA之后检测了IL-12p70的变化,结果显示转染miR-146a mimic过表达miR-146a后,较正常组CD11b缺陷小鼠DC组CpG-ODN刺激分泌的IL-12p70显著下调,而较CD11b缺陷小鼠DC正常小鼠DC转染miR-146a inhibitor后IL-12p70明显上调,可见CD11b的确通过辅助miR-146a的上调继而靶向Notch1来抑制了CpG-ODN触发的IL-12p70产生。
结语:
综上所述,CD11b参与、辅助了CpG-ODN诱导DC中miR-146a上调并由此靶向Notch1继而抑制了IL-12p70的产生,最终减弱了DC的抗原交叉提呈反应。本研究提供了整合素调控抗原交叉提呈反应的直接证据,并发现了miR-146a的新的生物学靶及其新功能,该结果不仅对CD11b调控miR-146a的机制进行了探讨分析,更为今后临床抗肿瘤、抗感染治疗中如何提升DC疫苗活性来增强抗原交叉提呈反应提供了新的靶点。
Immune activation or immune tolerance largely depends on the degree of antigen presentation and functional state of the immune cells involved. As specialized antigen-presenting cells and initiator of adaptive immune response, dendritic cells (DCs) can take up, process, and present exogenous antigens with MHC class II molecules to CD4~+ T cells. Apart from this, DCs can also present exogenous antigens to CTLs via MHC class I molecules. This special antigen presentation, known as cross-priming, has been shown to play critical roles in the immune defense against virus, bacteria, and tumor. During this process, IL-12p70, predominantly produced by DCs, not only enhances CTL synapse formation and prolongs CTL-DC interactions, but also promotes CTL proliferation and IFN-γproduction. DC cross-priming of CTL response is mediated by multiple mechanisms, such as activated costimulatory signals and attenuated proapoptotic signals. However, identifying positive and negative regulators in this process is still an ongoing process.
β2 integrins (CD11/CD18) play important roles in immune response and inflammation. According to their distinctαsubunits,β2 integrins are divided into four functional heterodimers termed leukocyte function-associated antigen 1 (LFA-1; CD11a/CD18,αLβ2 integrin, ITAL antigen), Mac-1 (CD11b/CD18,αMβ2 integrin, ITAM antigen), p150,95 (CD11c/CD18,αXβ2 integrin, CR4, ITAX antigen), andαDβ2 (CD11d/CD18, ITAD antigen). CD11b is extensively expressed in most immune cells, such as DC, monocytes, macrophages, granulocytes, and NK cells, participating in cell activation, chemotaxis, cytotoxicity, and phagocytosis. Our previous studies have shown that CD11b is able to maintain immunological tolerance and inhibit inflammatory responses via repressing TLR3 signaling in NK cells and TLR4 signaling in macrophages. Analogously, CD11b, abundantly expressed in DCs, can inhibit DC-primed CD4~+ T cell activation and promote peripheral tolerance by breaking Th17 differentiation. These reports have suggested the roles of CD11b in the regulation of T cell adaptive immunity. However, it remains unclear whether CD11b is also involved in DC cross-priming for CTL response. In CTL activation, it is well-accepted that TLR ligands, including LPS and CpG-ODN, are able to improve vaccination aiming at induction of CTLs, but the role of CD11b in this process is still unknown.
Although TLR4 and TLR9 both contribute to DC maturation and cytokine production, they depend on different signaling pathways. In DCs, LPS provokes cytokine production mainly through myeloid differentiation factor 88 (MyD88) dependent manner while induces upregulation of costimulatory molecules including CD40, CD80, and CD86 via the TIR domain-containing adaptor inducing IFN-β(TRIF) dependent manner. But TLR9 exclusively initiates a signaling cascade through MyD88, which activates NF-κB and MAPK pathways, and in turn induces proinflammatory cytokine production and DC maturation.
Micro-RNAs (miRNAs) are a class of small non-coding regulatory RNAs. They control gene expression at the post-transcriptional level by inhibiting translation or inducing mRNA degradation. miRNAs are emerging as important regulators in immune responses. Among them, miR-146a plays key roles in innate immunity, inflammatory response, virus infection, and human diseases. Both TLR activation and inflammatory stimulation can lead to upregulation of miR-146a, which in turn negatively regulates innate immunity by repressing its targets, such as IL1-R-associated kinase (IRAK) 1, IRAK2, TNFR-associated factor (TRAF) 6, and Tata Binding Protein (TBP). The induction of miR-146a is reported to be dependent on NF-κB activation, but the precise regulatory mechanism remains unclear. Apart from this, as a single miRNA has been thought to target multiple mRNAs, there are still many potential targets of miR-146a remain to be identified.
Here, in CD11b-deficient DCs, we found that TLR9-triggered IL-12p70 production was increased, and DC cross-priming for CTL response was elevated, suggesting the negative regulation of CD11b in DC cross-priming. For the mechanisms responsible for its negative regulation, we showed that CD11b participates in TLR9-mediated miR-146a upregulation in DCs. And the upregulated miR-146a in turn targets Notch1 to repress IL-12p70 production. Hence, our results reveal a novel role of CD11b in DC cross-priming for CTL response.
1. CD11b represses DC cross-priming of CTL response by inhibiting IL-12p70 production
To investigate the involvement of CD11b in DC cross-priming CTL response, we cultured BMDCs from wild-type or CD11b-deficient mice, and stimulated with TLR4 ligand (LPS) or TLR9 ligand (CpG-ODN) for 24 hours. When loaded with antigen OVA and co-cultured with na?ve CD8+ CTLs from OT-I mice, CD11b-deficient BMDCs had significantly enhanced CpG-triggered DC cross-priming of CTL response compared with that of wild-type BMDCs, as shown by more OVA-specific CTLs and higher IFN-γproduction. Unexpectedly, CD11b-deficiency did not affect LPS-triggered DC cross-priming CTL response. These results indicate that CD11b in DCs can negatively regulate TLR9, but not TLR4, -triggered cross-priming CTL response.
To further clarify the specific mechanism, we firstly detected the phenotype of CD11b-deficient DCs upon LPS or CpG-ODN stimulation. FACS analysis showed that CD11b-deficiency had little effect on the expression of MHC-I, MHC-II, CD40, CD70, CD80, or CD86 on both immature and mature BMDCs, thus indicating that CD11b mediated negative regulation of DC cross-priming CTL response was independent of cell surface costimulatory molecules. After screening of cytokines production in LPS or CpG-ODN stimulated DCs, we found that CD11b-deficiency had no significant influence on IL-6, TNF-α, or IL-1βproduction. Interestingly, CpG-ODN-induced IL-12p70 production was significantly enhanced in CD11b-deficient BMDCs than that in wild-type BMDCs, furthermore, specific neutralizing antibody to IL-12p70 diminished the differed CTL proliferation and IFN-γproduction between CD11b-deficient and wild-type DCs upon CpG-ODN stimulation. Because IL-12p70 is well-accepted to augment CTL activation, these results demonstrate that CD11b negatively regulates CpG-ODN-induced IL-12p70 production in DCs, and in turn represses DC cross-priming CTL response.
2. CD11b participates in CpG-ODN-induced miR-146a upregulation in DCs via sustaining late-phase NF-κB activation
In CpG-ODN-triggered DC cross-priming CTL response, CD11b only negatively regulates CpG-ODN-induced IL-12p70 production, but does not influence DC phenotype maturation or inflammatory cytokines production. This phenomenon indicates that CD11b is less likely to directly influence TLR9 signaling in DCs. So, some other indirect mechanisms may be responsible for the enhanced IL-12p70 production in CD11b-deficient DCs upon CpG-ODN stimulation.
miRNAs are important regulators in immune response. Thus, we wondered whether CD11b is involved in the regulation of miRNAs expression induced by CpG-ODN in DCs, which may subsequently contribute to the decreased IL-12p70 production. To further verify this hypothesis, we selected some miRNAs as candidates, especially those identified to be important regulators in TLR response. In details, miR-21 is known to regulate IL-12p35 expression, but CD11b-deficiency does not seem to affect miR-21 expression in LPS or CpG-ODN-stimulated DCs; miR-155 is another TLR signaling induced miRNA, and CD11b-deficiency has no significant effect on its expression either; for miR-146a, known to be upregulated upon TLR agonists stimulation, CD11b-deficiency inhibited CpG-ODN-induced miR-146a upregulation. Apart from this, in wild-type DCs, both LPS and CpG-ODN induced pri-miR-146a expression, and CD11b-deficiency also abolished CpG-ODN, but not LPS, -triggered pri-miR-146a expression, which suggests that miR-146a is regulated by CD11b at the transcriptional level. Since induction of miR-146a has been proved to be NF-κB dependent, we also validated that CpG-ODN-induced miR-146a expression was dependent on NF-κB activation, shown as NF-κB inhibitor inhibited its expression upon CpG-ODN stimulation in DCs. Furthermore, we determined the NF-κB activation by immunoblot, we found that CD11b doesn’t influence CpG-ODN-induced early-phase NF-κB activation, thus has no effect on proinflammatory cytokine production; but participates in CpG-ODN-induced late-phase NF-κB activation, and in turn upregulates miR-146a expression in DCs.
3. miR-146a represses CpG-ODN-induced IL-12p70 production by targeting Notch1 in DCs
As miRNAs function mainly through repressing their target mRNAs, and the known targets of miR-146a, such as IRAK1, IRAK2, TRAF6, and TBP, seem to be unrelated to IL-12p70 production, we searched new possible targets of miR-146a through TargetScan prediction (http://www.targetscan.org). Among the putative targets of miR-146a, we found that Notch1 had a putative conserved miR-146a target site. Notch signaling has been shown to be induced by TLR activation and participate in TLR activation-induced IL-12p70 production. To certify whether Notch1 3’UTR was directly targeted by miR-146a, we constructed a reporter plasmid containing Notch1 3’UTR and found that its expression was inhibited by miR-146a co-transfection, while the seed region mutated construct failed to be inhibited by miR-146a expression. This result suggests that Nortch1 is directly targeted by miR-146a expression.
In addition, in CpG-ODN-stimulated DCs, expression of full length Notch1 and Notch intracellular domain (NICD) 1 were increased by CD11b-deficiency, suggesting that CD11b may repress IL-12p70 production via inhibiting Notch1. To verify the impact of miR-146a on Notch1, we transfected miRNA into DCs, and miR-146a mimic markedly reduced CpG-ODN-induced Notch1 expression at both protein and mRNA levels, whereas miR-146a inhibitor increased their expression. Taken together, these results suggest that Notch1, as a new target of miR-146a, may be responsible for the downregulated IL-12p70 production by CD11b expression.
Next we examined the effects of Notch1 knockdown on CpG-ODN-triggered IL-12p70 production. Notch1 specific siRNA inhibited the expression of both full length Notch1 and NICD1, and Notch1 knockdown weakened IL-12p70 production in both wild-type and CD11b-deficient DCs upon CpG-ODN stimulation. These data suggests that Notch1 participates in CpG-ODN-triggered IL-12p70 production. We next determined whether miR-146a was responsible for the upregulated IL-12p70 production in CD11b-deficient DCs upon CpG-ODN stimulation. Transfection of miR-146a inhibitor promoted CpG-ODN induced IL-12p70 production in wild-type DCs but did not influence that in CD11b-deficient DCs. These data further confirm that CD11b represses CpG-ODN-triggered IL-12p70 production via upregulating miR-146a expression and targeting Notch1 in DCs.
Conclusion
Taken together, we demonstrate that CD11b is a negative regulator of TLR9 triggered DC cross-priming of CTL response via inhibiting IL-12p70 production in DC, which is mediated by upregulating miR-146a and targeting Notch1. Our study presents a new model in the negative regulation of TLR9 agonist-triggered DC cross-priming of CTL response by CD11b. Hence, CD11b and downstream miR-146a may be new regulators in DC licensed cross-priming aimed at induction of CTLs for the treatment of infectious diseases and cancer.
引文
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[31] Zhang T, Liu S, Yang P, et al. Fibronectin maintains survival of mouse natural killer (NK) cells via CD11b/Src/beta-catenin pathway[J]. Blood, 2009,114(19):4081-8.
[32] Banchereau J, Steinman RM. Dendritic cells and the control of immunity[J]. Nature, 1998,392(6673):245-52.
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[35] Lu TX, Munitz A, Rothenberg ME. MicroRNA-21 is up-regulated in allergic airway inflammation and regulates IL-12p35 expression[J]. J Immunol, 2009,182(8):4994-5002.
[36] O'Connell RM, Taganov KD, Boldin MP, et al. MicroRNA-155 is induced during the macrophage inflammatory response[J]. Proc Natl Acad Sci U S A, 2007,104(5):1604-9.
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[1] Shattil SJ, Kim C, Ginsberg MH. The final steps of integrin activation: the end game[J]. Nat Rev Mol Cell Biol, 2010,11(4):288-300.
[2] Abram CL, Lowell CA. The ins and outs of leukocyte integrin signaling[J]. Annu Rev Immunol, 2009,27:339-62.
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[5] Kim C, Lau TL, Ulmer TS, et al. Interactions of platelet integrin alphaIIb and beta3 transmembrane domains in mammalian cell membranes and their role in integrin activation[J]. Blood, 2009,113(19):4747-53.
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[1] Shattil SJ, Kim C, Ginsberg MH. The final steps of integrin activation: the end game[J]. Nat Rev Mol Cell Biol, 2010,11(4):288-300.
[2] Abram CL, Lowell CA. The ins and outs of leukocyte integrin signaling[J]. Annu Rev Immunol, 2009,27:339-62.
[3] Luo BH, Carman CV, Springer TA. Structural basis of integrin regulation and signaling[J]. Annu Rev Immunol, 2007,25:619-47.
[4] Xiong JP, Stehle T, Goodman SL, et al. New insights into the structural basis of integrin activation[J]. Blood, 2003,102(4):1155-9.
[5] Kim C, Lau TL, Ulmer TS, et al. Interactions of platelet integrin alphaIIb and beta3 transmembrane domains in mammalian cell membranes and their role in integrin activation[J]. Blood, 2009,113(19):4747-53.
[6] Evans R, Patzak I, Svensson L, et al. Integrins in immunity[J]. J Cell Sci, 2009,122(Pt 2):215-25.
[7] Avraamides CJ, Garmy-Susini B, Varner JA. Integrins in angiogenesis and lymphangiogenesis[J]. Nat Rev Cancer, 2008,8(8):604-17.
[8] Caswell PT, Norman JC. Integrin trafficking and the control of cell migration[J]. Traffic, 2006,7(1):14-21.
[9] Cahalan MD, Parker I. Choreography of cell motility and interaction dynamics imaged by two-photon microscopy in lymphoid organs[J]. Annu Rev Immunol, 2008,26:585-626.
[10] Zou Z, Chen H, Schmaier AA, et al. Structure-function analysis reveals discrete beta3 integrin inside-out and outside-in signaling pathways in platelets[J]. Blood, 2007,109(8):3284-90.
[11] Shtengel G, Galbraith JA, Galbraith CG, et al. Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure[J]. Proc Natl Acad Sci U S A, 2009,106(9):3125-30.
[12] Pribila JT, Quale AC, Mueller KL, et al. Integrins and T cell-mediated immunity[J]. Annu Rev Immunol, 2004,22:157-80.
[13] Depoil D, Fleire S, Treanor BL, et al. CD19 is essential for B cell activation by promoting B cell receptor-antigen microcluster formation in response to membrane-bound ligand[J]. Nat Immunol, 2008,9(1):63-72.
[14] Mocsai A, Abram CL, Jakus Z, et al. Integrin signaling in neutrophils and macrophages uses adaptors containing immunoreceptor tyrosine-based activationmotifs[J]. Nat Immunol, 2006,7(12):1326-33.
[15] Arnaout MA, Goodman SL, Xiong JP. Structure and mechanics of integrin-based cell adhesion[J]. Curr Opin Cell Biol, 2007,19(5):495-507.
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