Innate Immune Sensing of DNA

Author: Shruti Sharma, Katherine A. Fitzgerald*

When a pathogen attacks, the immune system rapidly mobilizes host defenses in order to reduce the microbial burden and limit damage to the host [1]. Innate immunity is the first line of defense and relies on germ line–encoded pattern recognition receptors (PRRs) such as the Toll-like receptors (TLRs), which sense microbial products that are not normally found on or in mammalian cells. The considerable potency of nucleic acids as triggers of the innate immune response has gained appreciation over the last few years. In particular, nucleic acid sensing of viruses is central to anti-viral defenses through recognition of viral genomes or nucleic acids generated during viral replication. Distinct classes of nucleic acid sensing molecules have been uncovered that function in different cell types and subcellular compartments to coordinate innate defenses (reviewed in [2]).

当病原体入侵时，免疫系统快速启动宿主免疫来降低微生物带来的压力并减少宿主伤害。天然免疫是抵御入侵的第一道防线，它依靠于种系遗传（高度保守）编码的模式识别受体（pattern recognition receptors, PRRs），如Toll样受体（TLRs），它们可以感知到在哺乳动物细胞中不会存在的微生物产物（“异己”产物）。过去几年，核酸作为天然免疫响应的激发原因的机制已经被不断解读。尤其是，在病毒在机体复制时，对于病毒核酸的感知是抗病毒防御中非常重要的一环。不同机制的核酸感知已经揭示了在不同细胞类型以及不同亚细胞组分的天然免疫过程。

While recognition of RNA molecules is dependent on members of the TLR family and cytosolic RNA helicases, the mechanisms underlying the sensing of DNA have been less well defined. It has been known for over a decade that DNA, the most recognizable unit of life, is a potent trigger of inflammatory responses in cells. The discovery of TLR-9, a receptor for hypomethylated CpG-rich DNA, partially explained these findings [3]. TLR9 is localized to the endosomal compartment and in humans is expressed in B cells as well as in plasmacytoid dendritic cells (pDCs). However, it became clear that the immune stimulatory activity of microbial DNA was not compromised in many cells lacking TLR9 [4]. These observations prompted new efforts to understand how DNA triggers immune responses, an endeavor that has led to the discovery of several new DNA recognition receptors and fresh insights into infectious as well as autoimmune diseases.

比起对TLR家族以及胞质RNA解旋酶对于RNA分子的识别机制，对于DNA感知的机制的了解要少很多。很多年以来，大家都清除，DNA这个生命最明显的特征，是细胞炎症反应中很可能的原因。人们发现了TLR-9，它可以作为去甲基化的富含CpG位点（CG位点）DNA的受体，这一发现佐证了以上想法。TLR9位于胞内体腔，在人类中，会在B细胞以及浆细胞样树突状细胞中表达。然而，人们也逐渐认识到，在很多并没有TLR9的细胞中，DNA天然免疫的活性也很高。这些发现更加支持了后续DNA如何激活天然免疫响应的研究，这也导向了全新的DNA识别受体的发现，也刷新了人们对天然免疫和病毒入侵的认知。

There Are Multiple Receptors for Microbial DNA

A significant effort from many laboratories has highlighted the importance of cytosolic DNA sensing in the innate immune response. At least six intracellular receptors have been implicated to some degree. These include DNA-dependent activator of interferon (IFN)-regulatory factors (DAI) (also called Z-DNAbinding protein 1, ZBP1) [5], absent in melanoma 2 (AIM2) [6–9], RNA polymerase III (Pol III) [10,11], leucine-rich repeat (in Flightless I) interacting protein-1 (Lrrfip1) [12], DExD/H box helicases (DHX9 and DHX36) [13], and most recently, the IFN inducible protein IFI16 [14]. DAI was the first to be implicated in synthetic B- and Z-form dsDNA recognition [5]; however, the role of DAI is still unclear, as DAI-deficient mice and cells coordinate normal immune responses to DNA [15]. Cytoplasmic dsDNA also triggers IFN production via RNA Pol III, which transcribes the DNA into 59-ppp RNA, a ligand for the RNA helicase RIG-I [10,11]. In pDCs, DHX9 and DHX36 contribute to cytosolic CpG-DNA and HSV-1-driven IFN responses [13], which likely account for previously reported TLR9-independent cytokine responses to some DNA viruses [12]. Lrrfip1 appears to bind both DNA and RNA; however, Lrrfip1 does not regulate the transcription factors that drive IFN gene transcription, but rather signals a co-activator pathway involving b-catenin and CBP/p300 histone modifying complexes to enhance the transcription of type I IFNs in the nucleus [16]. DNA from Listeria monocytogenes and RNA from vesicular stomatitis viral (VSV) activate this Lrrfip1-b-catenin pathway to mediate these effects.

胞质DNA感知在天然免疫中的重要性已然被揭示。至少6个胞内受体在不同程度上被揭示了。包括DNA依赖IFN激活体（DNA-dependent activator of interferon, DAI）——可调控因子，也被称作Z-DNA结合蛋白1（ZBP1）。以及“黑色素瘤中不存在（absent in melanoma 2 ）”——AIM2，以及RNA聚合酶III——Pol III，富含亮氨酸重复互作蛋白-1——Lrrfip1，DExD/H框解旋酶——DHX9和DHX36，以及最近发现的IFN促进型蛋白——IFI16。

……略（主要是介绍这6个DNA sensor的基本情况）

Immune responses to DNA are not restricted to type I IFNinducing pathways: cytosolic DNA also activates caspase-1dependent maturation of the pro-inflammatory cytokines interleukin (IL)-1b and IL-18. This pathway is mediated by AIM2, a PYHIN (Pyrin- and HIN200-domain-containing) protein. Recent evidence from knockout studies has revealed the importance of AIM2 in host defense to cytosolic bacteria such as Fransicella spp., as well as DNA viruses like mouse cytomegalovirus (reviewed in [17–20]). The newest receptor identified, IFI16, binds viral DNA and is critical in the immune response to certain DNA viruses [14]. Like AIM2, IFI16 is a PYHIN protein that binds viral DNA via HIN domains; however, IFI16 does not appear to associate with ASC to regulate IL-1b maturation. Rather, IFI16 activation induces IFN-b and inflammatory cytokine production in response to cytosolically administered viral DNA or HSV1 infection.

对于DNA的免疫应答不仅仅局限于IFN合成通路：胞内DNA也会激活预炎症因子白介素的胱天蛋白酶依赖型成熟（interleukin, IL）——IL-1b以及IL-18.这个通路又AIM2介导——一种PYHIN家族蛋白。最近研究表明，AIM2的敲除实验揭示了它在宿主免疫对胞质细菌的重要性。最新的DNA sensor IFI16，会结合病毒DNA并且在对特定DNA病毒的免疫反应中很重要。和AIM2一样，PYHIN蛋白会结合病毒DNA通过HIN域。然而IFI16并不会与ASC合作去调控IL-1b的成熟。另外，在胞内病毒DNA以及HSV1感染下，IFI16的活性促进IFN-b以及炎症因子的生成。

Distinct Classes of DNA Sensors Engage Distinct Signaling Complexes

Most of these DNA sensors utilize a subset of adapter molecules, which relay signals to NF-kB and members of the interferon regulatory factor (IRF) family. TLR9 as well as DHX9 and DHX36 recruit MyD88 to activate IFN production in pDCs in response to DNA. In contrast, recognition of DNA by RNA-Pol III generates an RNA intermediate, which signals via RIG-I and MAVS. In the case of IFI16, the endoplasmic reticulum–resident protein stimulator of interferon genes (STING) relays signaling downstream [21]. Whether STING binds IFI16 directly or merely acts as a signaling intermediate for this pathway is unclear. AIM2 triggers caspase-1 activation via the PYD domain containing adapter molecule ASC. Although IFI16 also contains a PYD domain, it does not appear to utilize ASC for IFN production. It is likely that the DAI pathway also involves STING, although this has not been formally demonstrated. Downstream of STING, MAVS, or MyD88, the nucleic acid sensing pathways converge on different IKK kinases to phosphorylate and activate IRFs (reviewed in [2]). In the case of the TLRs and possibly DHX helicases, IKKa is involved in phosphorylating IRF7, while downstream of MAVS and STING, TANK-binding kinase 1 (TBK-1), an IKK-related kinase, phosphorylates and activates IRF3. There is no evidence for the involvement of adaptor proteins in the Lrrfip1-b-catenin pathway, although intermediarysignaling molecules may be required for Lrrfip1-dependent bcatenin phosphorylation.

大多数DNA sensor都会利用一些可调控的分子集体，它们会把信号传递给NF-kB以及干扰素调控因子（IRF）家族。TLR9以及DHX9以及DHX36在DNA诱导下会招募MyD88在pDCs中去激活IIFN的合成。相反，RNA-Pol III对于DNA的识别会产生RNA中间体，通过RIG-I以及MAVS传递信号。对于IFI16，内质网定位的STING蛋白（stimulator of interferon genes）会把信号传递给下游。STING是会直接结合IFI16还是有其它分子作用目前还不清楚。

……略

Figure 1. Pathways of innate immune sensing of DNA. (A) Cytosolic DNA from invading viruses and bacteria engage and activate AIM2 binding to the adaptor ASC. ASC mediates caspase-1-dependent pro-IL-1b/pro-IL-18 cleavage and secretion of their bioactive forms, IL-1b and IL-18. IL-1b and IL-18 are significant mediators of inflammatory responses to infection. (B) Four known cytosolic sensors are represented here. Lrrfip1 recognized viral DNA as well as RNA to induce IFNb via a b-catenin-IRF3 transactivator pathway independently of the kinase TBK1. DAI can bind double-stranded B-form and atypical Z-form DNA to induce TBK1-IRF3-dependent IFNb production. Evidence for the role of adaptors MAVS/STING in these pathways is lacking. IFI16 can directly bind viral DNA via its HIN200 domains and initiate IFNb induction in a STING-TBK1- and IRF3-dependent manner. RNA polymerase III (Pol III) generates 59 tri-phosphate RNA that is a ligand for RIG-I. RIG-I signals via the adaptor MAVS, subsequently activating ubiquitin ligase TRAF3 and subsequently TBK1 and IRF3. The ubiquitin binding protein RNF5 inhibits STING activation by targeting it to the proteasome, while TREX1 inhibits/prevents IFNb production by degrading DNA substrate. (C) The receptor for advanced glycated end products (RAGE) and HMGB1 can bind extracellular CpG-rich DNA and transport it to a TLR9-positive compartment. Here, it is recognized by TLR9 and signals via MyD88 and the IKK kinase, IKKa, and IRF7 in pDCs to induce IFNa production. The cytosolic DExD/H box helicases DHX9/DHX36 can recognize cytosolic CpG DNA and initiate signaling to IRF7 via MyD88. 

图一 天然免疫中DNA感知的通路

A图：来自入侵病毒以及细菌的胞质DNA结合并激活AIM2结合调节因子ASC。ASC介导胱天蛋白酶1依赖型IL-1b以及IL-18的前体的切割以及分泌，IL-1b以及IL-18是重要的炎症反应的因子。

B图：四个已知胞质sensor在此处展示。

Lrrfip1识别病毒DNA以及RNA去促使IFN-b合成（通过b-连环蛋白-IRF3转录激活子通路）并依赖激酶TBK1.

DAI（ZBP-1）可以结合双链B型及Z型DNA去诱导TBK1-IRF3依赖型IFN-b的合成。在这通路里STING的作用还不清楚。

IFI16可以直接结合病毒DNA通过HIN200结构域，并且启动IFN-b的合成，通过STING-TBK1以及IRF-3依赖型机制。

……等等

本文一些观点比较老旧了，只做粗读，很多内容略过不做翻译了，作为笔者初学使用，希望广大看官不要介意啦~