Recognition of Cytosolic DNA
The innate immune system reacts to diverse molecules of microbial origin, termed pathogen-associated molecular patterns (PAMPs), or released from damaged or dying cells, called damage-associated molecular patterns (DAMPs). These molecules include nucleic acids, such as DNA. While the recognition of extra-cellular DNA involves mainly Toll-like receptor 9, recognition of cytosolic DNA appears to involve several sensors.
The first identified cytosolic DNA sensor, named DNAdependent activator of IFN-regulatory factors (DAI), binds cytosolic dsDNA and leads to the production of type I IFNs[1]. However, DAI defficiency does not affect the induction of type I IFNs in response to poly(dA:dT), a synthetic analog of B-DNA, suggesting that redundant cytosolic DNA sensors exist[2]. Unexpectedly, the next candidate receptor was the RNA helicase retinoic acidinducible gene-I (RIG-I), an RNA-binding and not DNAbinding protein. A human cell line deficient for RIG-I was shown to lack the ability to recognize poly(dA:dT)[3]. Recently, two independent teams confirmed the involvement of RIG-I in the response to dsDNA and demonstrated that rather than the cytosolic DNA, an RNA intermediate was responsible for RIG-I activation. They found that transfected poly(dA:dT) is transcribed by RNA polymerase III in the cytoplasm and potentially in the nucleus into a double-stranded RNA intermediate. This dsRNA molecule contains a 5’-triphosphate moiety and is recognized by RIG-I[4, 5]. Both DAI and RIG-I induce the production of type I IFNs through the TBK1/IRF3 pathway. The endoplasmic reticulum (ER)-resident transmembrane protein stimulator of IFN genes (STING) is a key component of this pathway[6]. STING seems to function as an adaptor protein upstream of TBK1.
Recently, a third IFN-inducer cytosolic dsDNA sensor has been identified[7]. This sensor LRRFIP1 can recognize ATrich B-form dsDNA as well as GC-rich Z-form dsDNA. With the use of LRRFIP1-specific siRNA, Yang et al. demonstrated that LRRFIP1 triggers the production of IFN-b in a b-catenindependent manner. b-Catenin binds to the C-terminal domain of IRF3 inducing an increase in IFN-b expression. Although the production of type I IFNs is the main pathway induced by cytosolic dsDNA, production of the pro-inflammatory cytokines IL-1b and IL-18 has also been observed. Recently, several groups have identify AIM2 (absent in melanoma 2), a member of the hematopoietic interferon-inducible nuclear protein HIN-200 family, as a cytosolic dsDNA sensor which activation promotes the assembly of an inflammasome[8-10]. DNA of various origins, such as poly(dA:dT), plasmidic DNA and DNA from the bacterium L. monocytogenes have been shown toactivate AIM2[11]. Upon activation, AIM2 interacts with ASC, a common adapter of the inflammasomes, leading to the cleavage of caspase-1 and the secretion of IL-1b and IL-18.
p202 is another member of the HIN200 family shown to bind cytoplasmic dsDNA but, in contrast to AIM2, it represses caspase activation[12]. The recognition of cytosolic DNA is more complicated than first anticipated. Several sensors have been identified that trigger different signaling pathways in a cell typespecific manner. Still, the general consensus is that another unknown cytosolic DNA-recognition system, independent of the TLRs and RIG-I, may exist. Further studies to elucidate the complex mechanisms of cytosolic DNA recognition may help the development of new strategies to treat nflammatory diseases.
InvivoGen provides an extensive set of tools to study the sensors of cytosolic double-stranded (ds)DNA.
These tools include synthetic analogs of dsDNA, a collection of human and mouse genes involved in the response to cytosolic dsDNA, CDS inhibitory molecules, and a reporter cell line.
Synthetic dsDNA Analogs - CDS Ligands
Poly(dA:dT) (B-DNA)
Poly(dA:dT) is a repetitive synthetic double-stranded DNA sequence of poly(dA-dT)•poly(dT-dA) and a synthetic analog of B-DNA. Poly(dA:dT) is recognized by several sensors, including DAI, LRRFIP1 and AIM2. It has also been shown to be transcribed by RNA polymerase III into dsRNA with a 5’-triphosphate moiety (5’ppp-dsRNA) which is a ligand for RIG-I.
Poly(dG:dC) (Z-DNA)
Poly(dG:dC) is a repetitive synthetic double-stranded DNA sequence of poly(dG-dC)•poly(dC-dG). Poly(dG:dC) is a synthetic analog of the Z-DNA form. It has been repor ted to be recognized by LRRFIP1.
Poly(dA:dT) and poly(dG:dC) are available naked or complexed with the cationic lipid LyoVec™ to facilitate their uptake. Their activity has been tested using the repor ter cell line B16-Blue™ IFNa/b (see below).
5’ppp-dsRNA
is a short (19 mer) blunt-end double-stranded RNA with a 5’ triphosphate. Transfected 5’ppp-dsRNA is a ligand of RIG-I. B16-Blue™ IFNa/b cells produce type I IFNs in response to transfected 5’ppp-dsRNA
pUNO plasmids - CDS Genes
CDS and CDS-related genes are provided in a pUNO plasmid which contains the complete coding sequence from the ATG to the Stop codon (www.invivogen.com/orfs).
Each gene is fully sequenced. pUNO plasmids are resistant to blasticidin.
BX795 and shRNAs - CDS Inhibitors
BX795
BX795, an aminopyrimidine compound, was developed as an inhibitor of 3-phosphoinositide-dependent kinase 1 (PDK1). It was recently shown to be a potent inhibitor of the IKK-related kinases, TANK-binding kinase 1 (TBK1) and IKKε, and hence of IRF3 activation and IFN-β production. BX795 inhibits the catalytic activity of TBK1/IKKε by blocking their phosphorylation.
shRNAs (Ready-made psiRNA plasmids)
shRNAs that target and silence by >70% CDS and CDS-related genes are expressed by ready-made psiRNA plasmids (www.invivogen.com/readymade-psirna). Their silencing efficiency is tested using the psiTEST system (www.invivogen.com/psitest-system).
B16-Blue™ IFN - IFNa/b Reporter Cell Line
B16-Blue™ IFN-a/b cells allow the detection of bioactive murine type I IFNs by monitoring the activation of the JAK/STAT/ISGF3 and/or IRF3 pathway. B16-Blue™ IFN-a/b cells derive from the murine B16 melanoma cell line of C57B1/6 origin. It was stably transfected with a SEAP reporter gene under the control of the IFN-a/b-inducible ISG54 promoter enhanced by five Interferon Stimulated Response Elements (ISRE).
Stimulation of B16-Blue™ IFN-a/b cells with murine IFN-a or IFN-b, or type I IFN inducers, such as tranfected poly(dA:dT) or 5’ppp-dsRNA, activates the JAK/STAT/ISGF3 and IRF3 pathways leading to the production of SEAP. Levels of SEAP in the supernatant can be easily determined with QUANTI-Blue™, a medium that turns purple/blue in the presence of SEAP and by reading the OD at 655 nm.
Ordering:
tlrl-patn, Poly(dA:dT) naked, 200ug, cena Volejte Kč
tlrl-patn-1, Poly(dA:dT) naked, 1mg, cena Volejte Kč
tlrl-patc, Poly(dA:dT) / LyoVec™, 100 ug, cena Volejte Kč
tlrl-pgcn, Poly(dG:dC) naked, 200 ug, cena Volejte Kč
tlrl-pgcc, Poly(dG:dC) / LyoVec™, 100 ug, cena Volejte Kč
tlrl-3prna-100, 5' ppp-dsRNA, 100 ug, cena Volejte Kč
puno-
tlrl-bx7, BX795, 5 mg, cena Volejte Kč
psirna42-
bb-ifnab, B16-Blue™ IFN- / cells, 5-7 x 10^6 cells, cena Volejte Kč
hkb-il1b, HEK-Blue™ IL-1 cells, 5-7 x 10^6 cells, cena Volejte Kč
References:
1. Takaoka A. et al., 2007. DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response. Nature. 448(7152):501-5.
2. Ishii KJ. et al., 2008. TANK-binding kinase-1 delineates innate and adaptive immune responses to DNA vaccines. Nature. 451(7179):725-9.
3. Cheng G. et al., 2007. Double-stranded DNA and double-stranded RNA induce a common antiviral signaling pathway in human cells. Proc Natl Acad Sci U S A. ;104(21):9035-40.
4. Ablasser A. et al., 2009. RIG-Idependent sensing of poly(dA:dT) through the induction of an RNA polymerase III-transcribed RNA intermediate. Nat Immunol. 10(10):1065-72.
5. Chiu YH. et al., 2009. RNA polymerase III detects cytosolic DNA and induces type I interferons through the RIG-I pathway. Cell. 138(3):576-91.
6. Ishikawa H. & Barber GN., 2008. STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling. Nature. 455(7213):674-8.
7. Yang P. et al., 2010. The cytosolic nucleic acid sensor LRRFIP1 mediates the production of type I interferon via a beta-catenindependent pathway. Nat Immunol. 11(6):487-94.
8. Hornung V. et al., 2009. AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC. Nature. 458(7237):514-8.
9. Fernandes-Alnemri T. et al., 2009. AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA. Nature. 458(7237):509-13.
10. Bürckstümmer T. et al., 2009. An orthogonal proteomic-genomic screen identifies AIM2 as a cytoplasmic DNA sensor for the inflammasome. Nat Immunol.10(3):266-72.
11. Jones Jw. et al., 2010.Absent in melanoma 2 is required for innate immune recognition of Francisella tularensis. PNAS, 107(21):9771-6.
12.Roberts TL. et al., 2009. HIN-200 proteins regulate caspase activation in response to foreign cytoplasmic DNA. Science ;323(5917):1057-60.
