Immune Sensing Mechanisms that Discriminate Self from Altered Self and Foreign Nucleic Acids

Abstract

All lifeforms have developed highly sophisticated systems equipped to detect altered self and non-self nucleic acids (NA). In vertebrates, NA-sensing receptors safeguard the integrity of the organism by detecting pathogens, dyshomeostasis and damage, and inducing appropriate responses to eliminate pathogens and reconstitute homeostasis. Effector mechanisms include i) immune signaling, ii) restriction of NA functions such as inhibition of mRNA translation, and iii) cell death pathways. An appropriate effector response is necessary for host defense, but dysregulated NA-sensing can lead to devastating autoimmune and autoinflammatory disease. Their inherent biochemical similarity renders the reliable distinction between self NA under homeostatic conditions and altered or exogenous NA particularly challenging. In this review, we provide an overview of recent progress in our understanding of the closely coordinated and regulated network of innate immune receptors, restriction factors, and nucleases to effectively respond to pathogens and maintain host integrity.

Figure 1

Principles of Self Versus Non-self or Altered-Self Nucleic Acid Recognition Unlike pathogen-specific molecules such as LPS, nucleic acids in pathogens and the host are biochemically similar. For a reliable distinction of self versus non-self and altered-self nucleic acid recognition, information about the molecular structure, the availability, and the localization is integrated. The localization of nucleic acid receptors on different cell types, immune cells as well as non-immune cells, contribute as well. Lists and cell types depicted are not comprehensive but just represent examples to better illustrate the principles.

Figure 2

RNA Sensing and Response Non-comprehensive overview of the most relevant RNA sensing receptors at the relevant localizations, with their downstream signaling molecules and some of the functional outcomes and secondary consequences as applicable (e.g., RIG-I sensing of RNaseL degradation products). RNA sensing receptors are in gray, signaling molecules in yellow, nucleases in green, inflammasome pathways is purple, and cell death pathways in orange. Unlike other TLRs, TLR3 signals from the cell surface and the endolysosome. TLR3 is a sequence-independent sensor of the ribose-phosphate backbone of dsRNA > 35bp and of incomplete dsRNA stem structures of sufficient length within ssRNA molecules. TLR3 signals via TRIF to induce IFN-β and NF-KB signaling. TLR7 and TLR8 are activated by RNA degradation products, with the first pocket binding guanosine and uridine (TLR7 and TLR8, respectively), and the second pocket binding short di- or trinucleotides. TLR8 activation requires upstream endosomal RNase activity (RNaseT2, RNase2) and, due to homology, RNase activity is likely required for TLR7 as well. TLR7 activation induces IFN-α via the MyD88-TRAF6-IRF7 pathway. TLR8 activation releases IFN-β and proinflammatory cytokines via a TAK1-IKKβ-IRF5 pathway. TLR adaptor interacting with SLC15A4 on the lysosome (TASL) was reported as a signaling component linking endolysosomal TLR7 and 8to IRF5. TLR13 recognizes bacterial 23S rRNA in a sequence-specific manner. Upon activation, the cytosolic immune sensors RIG-I and MDA-5 oligomerize thereby inducing polymerization of MAVS into fibrillar structures leading to the recruitment of TRAF2, TRAF6, IKK, and TBK1, which then activate NF-KB and IRF3 and/or IRF7 signaling. In addition, MAVS complexes can associate with FADD, RIP1, and caspase 8 and induce apoptosis downstream of MAVS. DDX3, DHX15, DHX36, and DDX60 enhance RIG-I signaling. DHX29 acts as co-receptor for both RIG-I and MDA5. LGP2 supports filament formation by MDA5 but may compete with RIG-I for ligand. Cytosolic RNA sensors with direct anti-viral activity include protein kinase R (PKR) and the 2’-5′-oligoadenylate synthetase system (OAS) which both bind dsRNA > 30bp including polyI:C. PKR phosphorylates elF2a and inhibits cap-dependent translation of viral and host mRNA. OAS induces the formation of 2′5′oligoadenylate which acts as a second messenger to activate ribonuclease L (RNase L), which in turn degrades cellular RNA and viral RNA to smaller RNA molecules that can be sensed by RIG-I and DHX33. DHX33 activates the NLRP3 inflammasome, inducing pyroptotic cell death. IFN-induced proteins with tetratricopeptide repeats (IFIT) sequester viral mRNA and block their translation by sensing 5′ termini. IFIT1 binds mRNA with a cap0 structure (7mGpppNN), and IFIT1B binds cap0, to a lesser extent cap1 (7mGpppNmN) structures but not cap2 (7mGpppNmNm) structures. b binds uncapped 5′ triphosphate RNA, and IFIT2 which binds AU-rich RNA. DDX17 can bind and sequester stem loop structures from some RNA viruses. Adenosine deaminase acting on RNA 1 (ADAR1) catalyzes the C6 deamination of adenosine to inosine in base-paired regions of RNA, and the resulting non-synonymous coding causes amino acid substitutions and potentially renders viral proteins non-functional. The host RNA decay machinery includes nonsense-mediated mRNA decay (NMD), 5′-3′ RNA degradation and the 3′-5′ RNA exonuclease machinery (RNA exosome). NMD targets mRNA transcripts with a long 3′ UTR but also senses viral RNA. The 5′-3′ degradation machinery with the decapping enzymes DCP1 and DCP2 and the 5′-3′ exonuclease XRN1 (XRN-DCPs) is involved in physiological cellular mRNA turnover and exhibits antiviral activity. The superkiller viralicidic activity 2-like (SKIV2L) and zinc-finger antiviral protein (ZAP) support the binding and transport vRNA to the RNA exosome for degradation. The ISG and viral restriction factor Z-DNA binding protein 1 (ZBP-1) is a death receptor downstream of dsRNA sensing. ZBP-1 activates multiple programmed cell death pathways, including pyroptosis, apoptosis, and necroptosis, termed PAN-optosis. Upon dsRNA binding, ZBP-1 interacts with RIPK3 supported by caspase-6, resulting in MLKL activation and necroptosis, in caspase-8-induced apoptosis and in NLRP3 dependent inflammasome activation and pyroptosis. Three inflammasome-building NLRs participate in cytosolic RNA sensing: DHX33 via NLRP3, the RNA helicase DHX9 via Nlrp9b (human homolog NLRP9), and the RNA-binding accessory protein DHX15, besides its supporting function for RIG-I-like helicases, via NLRP6.

Figure 3

DNA Sensing and Response Non-comprehensive overview of the most relevant DNA-sensing receptors at the relevant localization, their downstream signaling molecules, and the functional outcome and secondary consequences as applicable (e.g., STING sensing of cGAS product 2′3′cGAMP). DNA-sensing receptors are in gray, signaling molecules in yellow, nucleases in green, and inflammasome pathways is purple. The signaling competent form of TLR9 is exclusively localized to the endolysosome and preferentially detects ssDNA containing unmethylated cytosine-phosphate-guanine (CpG) motifs which are less frequent in eukaryotic self DNA compared to bacterial DNA. TLR9 has two binding sites, one site binding ssDNA with an unmethylated CpG motif and the second site binding short ssDNA carrying a 5′ hydroxyl, both cooperatively promoting TLR9 dimerization and signaling, including the recently identified signaling component TASL linking signaling to downstream IRF5. TLR9 ligands in the endolysosome are tightly regulated by endolysosomal DNase II, PLD3, and PLD4 which coordinately degrade single- and double-stranded DNA. Cytosolic DNA sensing includes the RNA sensor RIG-I which detects RNA transcribed from poly(dA:dT) by pol III. The cGAMP synthase (cGAS)/Stimulator of Interferon Genes (STING) pathway is the principle cytosolic dsDNA sensor pathway, activating a type-I IFN response, and via STING a NLRP3 dependent inflammasome response. Availability of cytosolic DNA for this pathway is regulated by DNase III (TREX1). Accessory proteins for cGAS are HMGB1, the GTPase-activating protein SH3 domain-binding protein 1 (G3BP1), TFAM, and CCHC-type zinc-finger protein 3 (ZCCHC3) which bind, bend, and stabilize dsDNA in a manner amenable to the nucleation of cGAS dimers along the dsDNA strand. cGAS is also localized in the nucleus where it senses foreign but not self DNA. Here, non-POU domain-containing octamer binding protein (NONO), but also HMGB1 and TFAM, assist in binding of DNA to cGAS. IFI16 is a nuclear restriction factor, binding, and silencing viral or transfected DNA. The DNA damage protein RAD50 upon stimulation with dsDNA induces proIL1β via the CARD9-BCL10 pathway. In the human system, a STING-independent DNA sensing pathway via the DNA damage response protein DNA-dependent protein kinase (DNA-PK) senses linear DNA, and signals via IRF3/IRF7 and HSPA8/HSC70. Absent in Melanoma (AIM2) is the principle cytosolic dsDNA sensor responsible for inflammasome activation. Several family members of the Apolipoprotein B editing complex (APOBEC) act as restriction factors by performing C-to-U editing on first(minus) strand cDNA, resulting in G-to-A mutations in the plus strand. The sterile alpha-motif (SAM) and histidine-aspartate (HD) domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase removing tripolyphosphate moieties from dNTPs thereby reducing the intracellular dNTP concentration and inhibiting reverse transcription.