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IMMUNOLOGY
1Laboratory of Human Retrovirology and Immunoinformatics,
Applied and Develop-mental Research Directorate, Leidos Biomedical
Research Inc., Frederick NationalLaboratory for Cancer Research,
Frederick,MD21702, USA. 2Laboratory of Proteomicsand Analytical
Technologies, Cancer Research Technology Program, Leidos
BiomedicalResearch Inc., Frederick National Laboratory for Cancer
Research, Frederick, MD21702, USA. 3Laboratory of Immunoregulation,
National Institute of Allergy andInfectious Diseases, National
Institutes of Health, Bethesda, MD 20892, USA.*Present address:
Inova Schar Cancer Institute, Inova Health System, Annandale,VA
22003, USA.†Corresponding author. Email: [email protected]
Sui et al., Sci. Signal. 10, eaah5054 (2017) 18 July 2017
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STING is an essential mediator of the Ku70-mediatedproduction of
IFN-l1 in response to exogenous DNAHongyan Sui,1 Ming Zhou,2*
Hiromi Imamichi,3 Xiaoli Jiao,1 Brad T. Sherman,1
H. Clifford Lane,3 Tomozumi Imamichi1†
We previously identified Ku70, a subunit of a DNA repair protein
complex, as a cytosolic DNA sensor that inducesthe production of
interferon-l1 (IFN-l1) by human primary cells and cell lines.
IFN-l1 is a type III IFN and hassimilar antiviral activity to that
of the type I IFNs (IFN-a and IFN-b). We observed that human
embryonic kidney(HEK) 293T cells, which are deficient in the innate
immune adaptor protein STING (stimulator of IFN genes), did
notproduce IFN-l1 in response to DNA unless they were reconstituted
with STING. Conversely, parental HEK 293 cellsproduced IFN-l1 after
they were exposed to exogenous DNA; however, when STING was knocked
out in the HEK293 cells through the CRISPR (clustered regularly
interspaced short palindromic repeats)/Cas9 genome editingsystem,
they lost this response. Through confocal microscopy, we
demonstrated that endogenous Ku70 was locatedin thenucleus and then
translocated to the cytoplasmuponDNAexposure to forma complexwith
STING.Additionally,the DNA binding domain of Ku70 was essential for
formation of the Ku70-STING complex. Knocking down STING inprimary
human macrophages inhibited their ability to produce IFN-l1 in
response to transfection with DNA or infec-tion with the DNA virus
HSV-2 (herpes simplex virus–2). Together, these data suggest that
STING mediates the Ku70-mediated IFN-l1 innate immune response to
exogenous DNA or DNA virus infection.
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INTRODUCTIONImmune recognition of pathogens is mediated by germ
line–encodedpattern recognition receptors (PRRs), which recognize
conserved mi-crobial structures termed pathogen-associated
molecular patterns(PAMPs) (1, 2). PRR families include the
Toll-like receptors (TLRs),the retinoic acid–inducible gene I
(RIG-I)–like receptors, and a diversefamily of cytosolic DNA
sensors (3–7). Once engaged by PAMPs, PRRsstimulate intracellular
signaling pathways and activate transcriptionfactors, such as
nuclear factor kB and interferon (IFN) regulatory factor3 (IRF3),
which lead to the increased production of proinflammatorycytokines,
such as tumor necrosis factor–a and interleukin-1b (IL-1b),and of
the antiviral type I IFNs, IFN-a or IFN-b (8, 9).
Considerable effort has beenmade to try to elucidate the initial
type IIFN signaling events that enable cells to detect the presence
of cytosolicDNA. This search has led to the identification
ofmultiple DNA sensors.The IFN-inducible protein DAI (DNA-dependent
activator of IRFs)was the first protein reported as a potential
mediator of the IFN responseto cytosolicDNA(10). LRRFIP1
(leucine-rich repeat flightless-interactingprotein 1) and ABCF1
(ATP-binding cassette, subfamily F member 1)also bind to DNA
directly and stimulate IFN responses (11, 12). TheAIM2 (absent in
melanoma 2)–like proteins, including human IFI16(IFN-g–inducible
protein 16) and its mouse ortholog protein p204,are also implicated
in DNA-mediated IFN responses (13–15). More-over, some DExD/H-box
helicases, including DDX41 (DEAD-box heli-case 41),DHX9
(DExH-boxhelicase 9), andDHX36 (DExH-boxhelicase36), are also
postulated to act as sensors of cytosolic DNA (16–18). Last,some
proteins with known functions in DNA damage responses are
mediators of the antiviral response triggered by cytosolic DNA.
Theseinclude components of the DNA-PK (DNA-dependent protein
kinase)complex, which is composed of Ku70, Ku80, and DNA-PKcs as
well asMRE11 (meiotic recombination 11 homolog A) (19, 20).
Type III IFNs are lesswell-characterized IFNswith a similar
antiviralactivity to that of the type I IFNs, and they include
IFN-l1, IFN-l2, andIFN-l3 (also known as IL-29, IL-28A, and IL-28B,
respectively) as wellas IFN-l4 (21–26). Expression of the genes
that encode IFN-l proteinsis inducible by infection with many types
of viruses (27–29). However,analysis of the murine genome showed
that the gene encoding themouse ortholog of human IFN-l1 lacks exon
2 entirely and containsa stop codon within exon 1 even in wild
mice; thus, the Ifnl1 gene doesnot encode a functional IFN-l1
protein (30). Therefore, any investiga-tion of the physiological
relevance of IFN-l1 in a mouse model isrestricted. Compared to the
signaling pathways associated with the pro-duction of type I IFNs,
the signaling pathways underlying the produc-tion of type III IFNs
are poorly understood.Wepreviously identified theDNA-PK component
Ku70 as a DNA sensor that stimulates type IIIIFN production by
human primary macrophages and human cell lines.Ku70, initially
characterized as a DNA repair protein, specifically bindsto
cytosolic DNA (such as after transfection) or viral DNA and
thenactivates the IFN transcription factors IRF1 and IRF7, leading
to robustproduction of IFN-l1 (31).Our previous study indicated
that the induc-tion of type III IFN production is distinct from
that of type I IFN pro-duction; however, the identity of the
adaptor protein downstream ofKu70was unknown.We also reported that
SV40T-antigen–transformedhuman embryonic kidney (HEK) 293 cells
(herein referred to as 293Tcells) donot produce IFN-l1 in response
toDNAstimulation.Themech-anism underlying this lack of response is
unknown (32).
The adaptor protein STING (stimulator of IFN genes) was
reportedto play a pivotal role in responding toDNAbymediatingTBK1
(TANKbinding kinase 1)–dependent activation of IRF3 in response to
cytosolic,double-stranded DNA (33–35). A wealth of information on
the role ofSTING inDNA sensing and on themechanisms whereby it
contributesto signaling in the induction of type I IFN production
has beendiscovered. In the signaling pathway to produce type I
IFNs, STINGacts
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downstream of several cytosolic DNA sensors, including DAI,
DDX41,cGAS [cyclic GMP (guanosine monophosphate)–AMP
(adenosinemonophosphate) synthase], and IFI16 (10, 15, 17, 36).
Other mediatorsinvolved in orchestration of the innate immune
response includeb-catenin, which functions downstream of LRRFIP1 to
stimulateIFN production, and the adaptor protein MyD88 (myeloid
differenti-ation primary response 88), which is involved in
responding to cyto-solic DNA through DHX9- or DHX36-mediated innate
immuneresponses (11, 16, 18). Here, we have looked for host
factor(s) involvedin the Ku70-mediated induction of type III IFN
production in re-sponse to exogenous DNA. Our findings offer a new
perspective onDNA sensing and have implications for host defense,
vaccine devel-opment, and autoimmunity.
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RESULTSAs compared to HEK 293 cells, 293T cells do not
produceIFN-l1 in response to DNAWe previously reported that Ku70 is
a cytosolic DNA sensor that in-duces IFN-l1 production by primary
human macrophages and somenonimmune cells, such as HEK 293 cells
and human rhabdo-myosarcoma cells (31), but not by 293T cells (32).
Here, we transfected293T cells and HEK 293 cells with plasmid
encoding green fluorescentprotein (GFP). One day later, the extent
of transfection was monitoredby fluorescence microscopy analysis.
Similar amounts of GFP were de-tected in both cell lines (Fig. 1A).
To determine the extent of expressionof the genes encoding IFNA,
IFNB, IFNL1, and IFNL2/3 in response tothe transfection, we
extracted cellular RNA from the transfected cellsand measured the
abundances of the appropriate mRNAs with areal-time reverse
transcription polymerase chain reaction (RT-PCR) as-say. Consistent
with our previous report (32), HEK 293 cells showed amore than
200-fold increase in the abundance of IFNL1 mRNA aftertransfection
with DNA. However, the abundances of IFNA, IFNB,and IFNL2/3mRNAs
were only 1.3-, 3.6-, and 61.3-fold greater, respec-tively,
compared to those in untreated cells (Fig. 1B). In contrast, none
ofthese mRNAs were increased in abundance in 293T cells after
transfec-tion with DNA (Fig. 1B). To further confirm induction of
the IFN re-sponse, we used enzyme-linked immunosorbent assays
(ELISAs) tomeasure the amounts of IFN-l1, IFN-a, IFN-b, and
IFN-l2/3 proteinsproduced by the cells. IFN-l1 was the major IFN
produced by the HEK293 cells, which had concentrations of 1393 ±
43.1 pg/ml in the culturemedium, whereas the concentrations of
IFN-l2/3, IFN-a, and IFN-bwere reduced at 371.0 ± 0.9, 61.3 ± 0.6,
and 157.3 ± 1.9 pg/ml, respectively(Fig. 1C).Consistentwith the
gene expressiondata,we failed to detect anyIFN-a, IFN-b, IFN-l1, or
IFN-l2/3 production by the transfected 293Tcells (Fig. 1C).
Additionally, a time course of the production of IFN-a,IFN-b,
IFN-l1, and IFN-l2/3 wasmeasured in the transfectedHEK cells(fig.
S1). On the basis of these data, we hypothesized that one or
moreimportant signaling factors were missing in the 293T cells.
293T cells lack endogenous STINGSTING is an importantmediator in
severalDNA-sensing pathways thatare associatedwith the induction of
type I IFN responses (10, 15, 17, 36).To determine whether
differences in the amounts of STING or Ku70might be associated with
the differential ability of 293T and HEK 293cells to produce
IFN-l1, we compared the amounts of STINGandKu70mRNAandprotein
between the two cell lines. The abundance of STINGmRNAwas less in
293T cells than inHEK293 cells (Fig. 2A). Consistentwith this
result, the abundance of STINGprotein, asmeasured byWest-
Sui et al., Sci. Signal. 10, eaah5054 (2017) 18 July 2017
ern blotting analysis, was alsomarkedly reduced in 293T cells
comparedto that in HEK 293 cells. There was no substantial
difference in theamount of Ku70 protein between 293T and HEK 293
cells (Fig. 2B).As additional controls, we also assessed the
amounts of b-catenin andMyD88 proteins in 293T andHEK 293 cells;
however, they were similarin both cases (fig. S2).
STING is an essential mediator in Ku70-mediated inductionof
IFN-l1 production in response to DNATo test the hypothesis that the
absence of STING in 293T cells wasresponsible for their inability
to produce IFN-l1 in response to DNA
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Fig. 1. Transfection of 293T cells with GFP-encoding plasmid DNA
does not in-duce the production of IFN-a, IFN-b, IFN-l1, or
IFN-l2/3mRNA and protein. (A toC) HEK 293 and 293T cells were
transfected with plasmid pCMV-GFP. (A) Twenty-fourhours later, the
cells were observed under a fluorescence microscope. Green
fluores-cence was shown in the context of total cells. Images are
representative of threeindependent experiments. (B) Total RNA was
extracted and the relative amounts ofIFNLA, IFNB, IFNL1, and
IFNL2/3mRNAsweremeasuredby real-timeRT-PCR. The relativeexpression
of the indicated genes was compared to that in untreated cells.
Data aremeans ± SDof three independent experiments. (C) Forty-eight
hours after transfection,the cell culturemediumwas collected, and
the concentrations of the indicatedproteinswere measured by ELISA.
Data are means ± SD of three independent experiments.
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exposure, we established an IFN-l1 reporter assay system. In
this sys-tem, 293T cells were cotransfected with pGL4–IFN-l1, a
reporter plas-mid containing the IFNL1 promoter region, and
different amounts ofplasmid encoding Ku70 with or without 100 ng of
plasmid encodingSTING. To fully induce Ku70 and STING, the
transfected cells wereincubated for 48 hours andwere then further
stimulated by being trans-fected with noncoding plasmidDNA, and
IFNL1 promoter activity wasquantitated by relative luciferase
activity (Fig. 3A).We found that Ku70induced IFNL1 promoter
activity only in the presence of STING, and itdid so in a
concentration-dependentmanner. Note that Ku70 alonewasnot able to
induce IFNL1 promoter activity. We detected substantialamounts of
STING in those 293T cells that were transfected with
theSTING-encoding plasmid (Fig. 3B); however, STING was
undetectablein 293T cells that were transfected with an empty
vector. To furtherconfirm the effect of exogenous STING in 293T
cells on IFNL1 expres-sion, we performed real-timeRT-PCR assay
using cellular RNA isolatedfrom DNA-stimulated,
STING-overexpressing 293T cells. The abun-dance of IFNL1 mRNA in
the STING-expressing 293T cells wasincreased in response to DNA
stimulation (Fig. 3C), whereas 293T cellsfailed to induce IFNL1
expression if theywere transfectedwith an emptyplasmid,whichwas
similar to the IFNL1 response observed in untreated293T cells (Fig.
3C). We next investigated whether the removal ofSTING from HEK 293
cells would modify the DNA-stimulated induc-tion of IFNL1
expression. For these experiments, STING knockout(STING KO) HEK 293
cells were generated through the clustered reg-
Sui et al., Sci. Signal. 10, eaah5054 (2017) 18 July 2017
ularly interspaced short palindromic repeats (CRISPR)/Cas9
genomeediting system. STING was undetectable in the STING KO cells;
how-ever, it was detectable after the cells were transfected with a
STING ex-pression plasmid (Fig. 3D). The abundance of Ku70was
similar in all ofthe cell lines examined, suggesting that the loss
of STING had no effecton Ku70 abundance. STINGKO cells did not
exhibit IFNL1 expressionafter transfection with DNA. However, the
DNA-stimulated inductionof IFNL1 expression was restored in cells
transfected with the STING-encoding plasmid (Fig. 3E). Together,
the results suggest that STING isan essential mediator of the
DNA-stimulated induction of IFNL1.
To exclude the possibility that the role of STING in
facilitatingIFN-l1production in response toDNAwas cell
type–dependent,weper-formed similar experiments with the THP-1 cell
line. THP-1 cells arehuman leukemia monocytic cells, which have
been extensively used tostudymonocyte andmacrophage functions,
mechanisms, and signalingpathways as well as nutrient and drug
transport (37). Western blottinganalysis confirmed that THP-1 cells
have endogenous Ku70 andSTING. Transfection of the cells with small
interfering RNAs (siRNAs)specific for Ku70 and STING reduced the
abundances of the target pro-teins by 51 and 66%, respectively,
compared to those in cells transfectedwith control siRNA (Fig. 3F).
Using the cells in which Ku70 or STINGwere knocked down, we further
performed transfections with plasmidDNA and assessed the expression
of IFN-l1. Similar to HEK 293 cells,THP-1 cells exhibited a
substantial (12,131-fold) increase in IFNL1mRNA in response to
transfection with DNA; however, this inductionwas inhibited in
cells in which the abundances of either Ku70 or STINGwere knocked
downby 59 and 78%, respectively (Fig. 3G). In cells trans-fected
with both siRNA-Ku70 and siRNA-STING, the induction ofIFNL1
expression was inhibited by 88% compared with that in the cellsthat
were not exposed to siRNA (Fig. 3G). Consistent with these data,the
abundance of IFN-l1 protein in the THP-1 cells in which the
abun-dance of Ku70 and STINGwas reduced by 96% compared to that in
thecells that were not exposed to siRNA (Fig. 3H), suggesting that
theKu70-STING pathway plays a role in the DNA-stimulated
inductionof IFN-l1 production in THP-1 cells (Fig. 3, G andH). The
knockdownof Ku70 and STING in the THP-1 cells also suppressed the
productionof IFN-a, IFN-b, and IFN-l2/3 (fig. S3).
We previously reported that cross-talk between signaling by
theDNAsensor IFI16 and the RNA sensor RIG-I mediates the type III
IFN re-sponse. Here, we investigated whether IFI16 was also
involved in theKu70-mediated production of IFN-l1 in response to
exogenous DNA.We knocked down IFI16 by transfecting the cells with
IFI16-specificsiRNA and confirmed the efficiency of knockdown
byWestern blottinganalysis (fig. S4A). However, the extent of IFNL1
induction was notchanged in the IFI16-knockdown cells compared with
that in the cellsthat were not treated with siRNA (fig. S4B).
Ku70 translocates from the nucleus to the cytoplasm andforms a
complex with STING in response to DNAOur findings led us to
investigate the physical relationship betweenSTING and Ku70. The
locations of endogenous Ku70 and STINGwereanalyzed by
immunofluorescence staining and confocal microscopyanalysis of
unstimulated and DNA-stimulated cells (Fig. 4A). Thecellular
localization was verified by counterstaining nuclei with
4′,6-diamidino-2-phenylindole (DAPI). In unstimulated cells, Ku70
wasdetected in the nucleus, whereas STING was found in the
cytoplasm(Fig. 4A, top). After the cells were stimulatedwithDNA,we
found thatKu70 translocated from the nucleus to the cytoplasm,
whereas STINGremained in the cytoplasm (Fig. 4A, bottom). This
translocation of
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Fig. 2. Comparison of the amounts of endogenous STING and Ku70
betweenHEK 293 cells and 293T cells. (A) Total RNA was extracted
from HEK 293 and 293Tcells, and the relative amounts of STING and
KU70mRNAs weremeasured by real-timeRT-PCR. The amounts of the
indicated mRNAs were normalized to that of GAPDHmRNA. Data are
means ± SD of three independent experiments. (B) Whole-cell
lysatesof HEK 293 and 293T cells were analyzed by Western blotting
with antibodies againstSTING and Ku70. b-Actin was used as a
loading control. Western blots are representa-tive of three
independent experiments.
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Ku70 from the nucleus to the cytoplasm provided an opportunity
forSTING and Ku70 to interact with each other.
We further designed a coimmunoprecipitation assay to
confirmwhether STING interacted with Ku70 in the context of
stimulation withDNA. The results demonstrated that in the absence
of exogenousDNA,FLAG-tagged Ku70 did not interact with exogenous
STING. However,Flag-tagged Ku70 coimmunoprecipitated with STING
when the cellswere treated with DNA (Fig. 4B). We also performed
the coimmuno-precipitation assay with an anti-Myc antibody to pull
down STING(Fig. 4C). The results of these experiments consistently
supported thehypothesis that STING and Ku70 are found in the same
complex onlyafter the cells are transfected with DNA (Fig. 4, B and
C).
Sui et al., Sci. Signal. 10, eaah5054 (2017) 18 July 2017
The DNA binding domain of Ku70 is required for Ku70 toform a
complex containing STINGKu70 is composed of an a/b domain (I), a
b-barrel DNA binding do-main (II), a linker (III), and a C-terminal
domain (IV) (Fig. 5A). Toanalyze which domain is involved in the
binding of Ku70 to STING,we constructed several truncated,
Flag-tagged Ku70 mutants, contain-ing the I, II, and III domains
(I.II.III), the I and II domains (I.II), orthe I domain alone (I).
We then performed coimmunoprecipitation as-says with 293T cells
transfected with plasmids encoding the Flag-taggedKu70mutants and
detectedwhich of these bound to STING in responseto DNA.We found
that Ku70 mutants with I.II.III.IV, I.II.III, I.II, or II.III.IV
coimmunoprecipitated with STING; however, those Ku70 mu-tants with
I.III.IV, I.III, I, or III.IV did not (Fig. 5B). Thus, it
appearedthat theDNAbinding domain (II) was required for the binding
of Ku70to STING. To confirm this finding, we constructed a
Ku70mutant con-sisting of only the DNA binding domain (Ku70 mutant
II). We foundthat this protein domain coimmunoprecipitated with
STING (Fig. 5B),confirming the importance of this region of Ku70 in
binding to STING.
A heterodimer of Ku proteins (Ku70 or Ku80) and the catalytic
sub-unit DNA-PKcs are the components of the DNA-PK complex. We
ad-ditionally confirmed whether Ku80 andDNA-PKcs were present in
the
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Fig. 3. STING is critical for the DNA-stimulated, Ku70-mediated
induction ofIFN-l1 production. (A) 293T cells were cotransfected
with an IFNL1 reporter plasmid(pGL4–IFN-l1), a Renilla luciferase
plasmid (pRL-TK), and different amounts of theKu70 expression
plasmidwith or without transfectionwith 100 ng of the
STINGexpres-sion plasmid, as indicated. Forty-eight hours after
transfection, the cellswere then stim-ulated with 1 mg of noncoding
plasmid DNA. Twenty-four hours after stimulation, thecells were
collected for luciferase assays. Data are means ± SD of three
independentexperiments. (B) Western blotting analysis was performed
to confirm the presence ofSTING in the transfected 293T cells.
Untreated 293T cells and 293T cells transfectedwith empty plasmid
were included as controls. Whole-cell lysates were analyzed
withanti-STING and anti-Ku70 antibodies. b-Actin was detected as a
loading control. West-ern blots are representative of three
independent experiments. (C) 293T cells were leftuntreated or were
transfected with either an empty plasmid or a STING
expressionplasmid and then were stimulated with 1 mg of plasmid
DNA. One day later, IFNL1mRNA abundances were measured by real-time
RT-PCR. Relative values weredetermined by comparison with untreated
293T cells. Data are means ± SD of threeindependent experiments.
**P < 0.001. (D) Western blotting analysis was performed
toconfirm the knockout of STING in the STING KO cells. Whole-cell
lysates were analyzedwith anti-STING and anti-Ku70 antibodies.
b-Actin was measured as a loading control.Western blots are
representative of three independent experiments. (E) STING KO
cellswere transfectedwith an empty vector or a STINGexpression
vector, and the cells werethen stimulated by transfection with 1 mg
of noncoding plasmid DNA. One day later,total cellular RNA was
extracted and the relative abundance of IFNL1 mRNA wasmeasured. The
values were compared to the amount of IFNL1mRNA in HEK cells
thatwere transfected with empty vector without additional DNA
stimulation. Data aremeans ± SD of three independent experiments.
**P < 0.001. (F) Western blottinganalysis was performed to
confirm the knockdown of Ku70 or STING in THP-1 cells.THP-1 cells
were transfected with or without the indicated siRNAs and then
weretransfected with DNA 1 day later. Whole-cell lysates were
collected and incubatedwith anti-STING and anti-Ku70 antibodies.
b-Actin was used as a loading control.Western blots are
representative of three independent experiments. (G)
Twenty-fourhours after the indicated THP-1 cells were stimulated by
transfection with DNA, theabundance of IFNL1mRNA was measured by
real-time RT-PCR. Relative values weredetermined by comparison with
untreated THP-1 cells. Data are means ± SD of threeindependent
experiments. (H) Forty-eight hours after the indicated THP-1 cells
werestimulated by transfection with DNA, the cell culture medium
was collected and theamounts of IFN-l1 were determined by ELISA.
Data are means ± SD of threeindependent experiments.
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Ku70-STING complex. The proteins that coimmunoprecipitated
withthe DNA binding domain of Ku70 were analyzed by Western
blottingwith anti–DNA-PKcs, anti-STING, and anti-Ku80 antibodies,
and theresult showed that Ku80was present in the complex but
thatDNA-PKcswasnotnecessarily required for the interactionof
theDNAbindingdomainof Ku70 with STING (fig. S5A). An IFNL1 promoter
reporter assayfurther suggested that the DNA binding domain alone,
like full-lengthKu70, dose-dependently activated IFNL1 promoter
activity (fig. S5B).Ku80, even if present in the Ku70-STING
complex, was not functionalin the DNA-mediated induction of IFN-l1
production (fig. S5B).
The activation of the transcription factors IRF3, IRF1, andIRF7
is required for the Ku70- and STING-mediatedproduction of IFN-l1 in
response to exogenous DNAHaving identified STING as a critical
protein in the Ku70-mediatedproduction of IFN-l1, we then
determined which transcription factor(s)was involved. First, we
tested the effect of knocking down IRF1, IRF3,or IRF7 on the
induction of IFN-encoding genes.We transfectedHEK293 cells with
siRNAs targeting IRF1, IRF3, or IRF7. On the following
Sui et al., Sci. Signal. 10, eaah5054 (2017) 18 July 2017
day, the cells were then transfected withDNA. Whole-cell lysates
were then col-lected and analyzed by Western blottingto determine
IRF1, IRF3, and IRF7 abun-dance, and total RNA was extracted
tomeasure the relative amounts of IFNmRNAs by real-time RT-PCR
analysis.In the absence of exogenous DNA, theabundance of IRF3 in
HEK 293 cellswas greater than that of either IRF1 orIRF7 (Fig. 6A).
In response to transfectionwith DNA, the amounts of IRF1 andIRF7,
but not that of IRF3, were increased(Fig. 6A). Transfection of the
cells withIRF1-specific siRNA not only efficientlyreduced the
abundance of IRF1 but alsodecreased that of IRF7 (Fig. 6A). In
addi-tion, the abundances of both IRF7 andIRF1 were reduced in
cells transfectedwith IRF7-specific siRNA. Furthermore,transfection
of the cells with IRF3-specificsiRNAnot only reduced the abundance
ofIRF3 but also reduced the amounts ofIRF1 and IRF7 (Fig. 6A). Our
real-timeRT-PCR analysis showed that the induc-tion of IFNL1
expression and IFN-l1pro-tein production were inhibited in cells
inwhich IRF1, IRF3, or IRF7 was knockeddown (Fig. 6, B andC).
Similar resultswereshown for IFN-a, IFN-b, and IFN-l2/3(fig. S6, A
and B).
Given that IRF3 and IRF7 reside in thecytosol and undergo serine
phosphoryl-ation of their C-terminal regions in re-sponse to viral
infection, which enablesthem to dimerize and translocate to
thenucleus (38), we next evaluated the nucle-ar accumulation of
IRF1, IRF3, and IRF7in HEK 293 cells and STING KO cells inresponse
to stimulation DNA. We trans-
fected the cells withDNAand thenprepared nuclear fractions at
varioustimes (Fig. 6D). Western blotting analysis showed that the
abundancesof IRF1 and IRF7, but not that of IRF3, were increased in
the nucleusof HEK 293 cells 48 hours after transfection. In
contrast, no increase inthe nuclear accumulation of these
transcription factors was observed inthe STINGKO cells (Fig. 6D).
The results demonstrated that the DNA-dependent activation of IRF1
and IRF7 occurred only in the presence ofSTING.
To further elucidate the role of STING in the activation of IRF1
andIRF7 after transfection of cells with DNA, we used confocal
microscopyto monitor the nuclear translocation of endogenous IRF1
and IRF7 inHEK 293 and STING KO cells (Fig. 6, E to H). Forty-eight
hours aftertransfection of the cells with DNA, we observed
substantial accumula-tion of both IRF1 and IRF7 in the nuclei of
HEK 293 cells (Fig. 6, E andG). Similar changes were not observed
in the STING KO cells (Fig. 6, Fand H). These results are
consistent with STING playing a key role inactivating these
transcription factors upstream of IFN gene induction.In summary,
the absence of STING dampened the activation of IRF1and IRF7 and
consequently resulted in reduced IFN-l1 production.
DAPI Ku70 STING Merge
Untreated
DNA-treated
A
IP-FlagFlag
STING
Myc-STING
Flag-Ku70
DNA
Flag
STING
Input
IP-MycSTING
Ku70
Flag-Ku70
Myc-STING
DNA
STING
Ku70
Input
B C
Fig. 4. STING and Ku70 colocalize in the cytoplasm and form a
protein-protein complex after cells are transfectedwith DNA. (A)
Confocal microscopy analysis of the localization of Ku70 and STING
in HEK 293 cells. HEK 293 cells weregrown on glass-coated, 35-mm
culture dishes and mock-transfected or transfected with 1 mg of
plasmid DNA. Twenty-four hours later, the cells were fixed and
stained with anti-Ku70 (green) and anti-STING (red) antibodies.
Nuclei werevisualized by DAPI (blue). Original magnification was
×60. Scale bars, 5 mm. Images are representative of
threeindependent experiments. (B and C) 293T cells were transfected
with the indicated combinations of plasmids encodingFlag-tagged
Ku70 and Myc-tagged STING and were stimulated by transfection with
DNA on day 2. On day 3, cytosoliclysates were subjected to
immunoprecipitation (IP) with agarose conjugated with anti-Flag
antibody (B) or anti-Mycantibody (C). Precipitatedproteinswere then
analyzed byWesternblottingwith antibodies against the indicated
targets.Input controls were also included. Western blots are
representative of three independent experiments.
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Loss of STING inhibits IFN-l1 production by human
primarymacrophages in response to transfection with DNA orinfection
with herpes simplex virus–2To further demonstrate the physiologic
relevance of STING in theKu70-mediated production of IFN-l1 in
response to DNA or DNAvirus, we transduced human monocyte-derived
macrophages (MDMs)with lentiviruses encoding short hairpin RNA
(shRNA) targeting theSTING expression cassette. The transduced
cells were then transfected
Sui et al., Sci. Signal. 10, eaah5054 (2017) 18 July 2017
with DNA plasmids or infected with her-pes simplex virus–2
(HSV-2). Twenty-four or 48 hours later, the cells werecollected to
analyze gene expression, andthe culture medium was harvested
tomeasure the abundances of IFN proteins.We first showed that
transduction withthe lentivirus led to a substantial reduc-tion in
the abundance of STING protein;however, no changes in STING
abun-dance were observed in untransducedcells or in cells
transduced with lentivirusencoding control shRNA (Fig. 7A).
Wefurther found that theDNA-increased ex-pression of IFNL1 was
inhibited by up to80% when STING was knocked down byshRNA (Fig.
7B). Analysis of IFN-l1 pro-tein abundance in the cell
culturemediumgave consistent results (Fig. 7C).
In addition, we also evaluated the effectof knocking down STING
on IFN-l1 pro-duction in cells infected with HSV-2. Theresults of
this experiment showed thatknocking down STING resulted in a
re-duction in the amounts of IFNL1 mRNAandprotein (Fig. 7,DandE).
Furthermore,knocking down STING also suppressedthe induction of
IFNA, IFNB , andIFNL2/3 (fig. S7). Together, these data sug-gest
that STING mediates the Ku70-dependent production of IFN in
responseto exogenous DNA or infection with aDNA virus.
DISCUSSIONThe innate immune system is the firstline of defense
against invading patho-gens. It is well known that microbial
nu-cleic acids stimulate the production oftype I IFNs, such as
IFN-a and IFN-b,as a key host defense strategy to limitthe
replication of invading microorga-nisms (39). Many of these
pathways andtheir capacity to induce type I IFN pro-duction have
been extensively studied(40). Accumulated evidence suggests
thatnon-self nucleic acids can also stimulatea type III IFN
response (21, 23, 31, 32);however, the molecular mechanismsinvolved
are poorly understood.
The DNA repair protein Ku70 also acts as a DNA sensor to
inducethe production of IFN-l1, as opposed to IFN-a or IFN-b, in
primarycells or cell lines, such as HEK 293 cells, but not 293T
cells (31, 32).However, the downstream mediator of Ku70 was
unclear. Here, bycomparing the properties of HEK 293 and 293T
cells, we identifiedSTING, located downstream of Ku70, as an
essential mediator of theproduction of IFN-l1 in response to
exogenous DNA. The inabilityof 293T cells to produce IFN-l1 after
transfection with DNA was
A
I: α/β domain (24.04 kDa) II: β-Barrel DNA-binding (31.38
kDa)III: Linker (8.69 kDa) IV: C-terminal (5.80 kDa)
Flag5'UTR
Ku70 (WT) I II III IV
STING
STING
Flag
Flag
IP-Flag
Input
Ku70 mutants:
B
Fig. 5. The DNA binding domain of Ku70 is essential for its
ability to form a complex with STING. (A) Schematicrepresentation
of the domain organization of Ku70. The molecular mass of each
domain of Ku70 is labeled. WT, wild type.(B) 293T cells were
transfected with plasmids encoding the indicated Flag-tagged
truncated Ku70 constructs and Myc-tagged STING andwere then
stimulated by transfectionwith DNA on day 2. One day later,
cytosolic lysates were subjectedto immunoprecipitation with
anti-Flag antibody–conjugated agarose. Precipitated proteins were
analyzed by Westernblotting with anti-Flag and anti-STING
antibodies. Input controls were also included. Western blots are
representative ofthree independent experiments.
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0
50
100
150
200
250
Rel
ati
ve
IFN
L1 m
RN
A
ab
un
da
nce
0
500
1000
1500
IFN
-λ1
, p
g/m
l
A
B C
D
IRF7 (65 kDa)
IRF1 (45~48 kDa)
NUP98 (98 kDa)
0 24 48 0 24 48
DNA transfection, hours
HEK 293 STING KO
IRF3 (45~55 kDa)
24 24
E
DAPI IRF1 Merge
Untreated
G
Untreated
DAPI IRF1 Merge
F
H
Untreated
IRF7 MergeDAPI
Untreated
DAPI IRF7 Merge
IRF3
IRF7
β-Actin
IRF1
DNA transfection, hours
DNA 48 hours DNA 48 hours
DNA 48 hours DNA 48 hours
Sui et al., Sci. Signal. 10, eaah5054 (2017) 18 July 2017
Fig. 6. The activation of IRF1, IRF3, and IRF7involves the
Ku70-mediated induction ofIFN-l1 production in response to
exogenousDNA. (A) HEK 293 cells were left untreated orwere
transfected with the indicated siRNAs.The cells were then
transfected with DNA1 day later. Twenty-four hours after
transfectionwith DNA, whole-cell lysates were analyzed byWestern
blotting with antibodies against the in-dicated proteins. Untreated
cells were includedas a control, and b-actin was used as a
loadingcontrol for each condition.Western blots are rep-resentative
of three independent experiments.(B) Total RNA was extracted 24
hours after DNAtransfection, and the relative abundance ofIFNL1
mRNA was measured by real-time RT-PCR. The relative values were
determined bycomparison with untreated cells. Data aremeans ± SD of
three independent experiments.(C) Forty-eight hours after DNA
transfection, thecell culture medium was collected and theamounts
of IFN-l1 protein were detected byELISA. Data are means ± SD of
three inde-pendent experiments. (D) HEK 293 and STINGKO cells were
stimulated by transfection with1 mg of plasmid DNA. Nuclear
fractions of thecells were isolated at the indicated times
aftertransfection and were analyzed by Westernblotting with
antibodies against the indicatedproteins. NUP98 was measured as a
loadingcontrol. Western blots are representative ofthree
independent experiments. (E to H) HEK293 and STING KO cells were
grown on glass-coated, 35-mm culture dishes. The cells
weretransfected with plasmid DNA. Forty-eighthours later, the cells
were fixed and stainedwithantibodies against IRF1 (green) (E and F)
or IRF7(red) (G and H) and visualized by confocal mi-croscopy.
Nuclei were visualized with DAPI(blue). Scale bars, 5 mm. Original
magnificationwas ×60. Images are representative of threeindependent
experiments.
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reversed by expression of the adaptor protein STING. In
contrast, lossof STING inhibited the DNA-induced production of
IFN-l1 by HEK293 cells. Additionally, we further clarified that
this finding was notrestricted to HEK 293 cells by showing that the
Ku70-STING pathwaywas required to mediate DNA-induced IFN-l1
production by THP-1cells and primary human macrophages.
Many studies about innate immune responses are performed
withmice deficient in the protein of interest. However, in the case
of IFN-l1,the mouse Ifnl1 gene lacks exon 2 and contains a stop
codon withinexon 1 even in wild mice, which results in the failure
to encode a func-tional IFN-l1 protein (30). Therefore, to
demonstrate the physiologicalrelevance of STING in the
Ku70-mediated production of IFN-l1 in re-sponse toDNA, we used a
lentiviral approach to knock down STING inhuman primary
macrophages. We found that the loss of STINGmark-edly inhibited the
generation of IFNL1mRNA and protein in responseto transfection with
DNA or infection with a DNA virus.
Sui et al., Sci. Signal. 10, eaah5054 (2017) 18 July 2017
Confocal microscopy analysis showed that Ku70 was found in
thenuclei of unstimulated cells. We found that Ku70 underwent a
trans-location from the nucleus to the cytoplasm when the cells
were trans-fected with a DNA plasmid. In contrast, STING was
located in thecytoplasm in both unstimulated and stimulated cells.
These results sug-gested that the translocation of Ku70 prompted an
interaction betweenKu70 and STING. Further work is necessary to
uncover the mecha-nisms about the translocation of Ku70. In
addition, coimmunoprecipi-tation experiments demonstrated that Ku70
formed a complex withSTING in response to transfection with DNA.
Ku70 is a componentof the heterotrimeric protein complex DNA-PK,
which also containsKu80 and the catalytic subunit DNA-PKcs. DNA-PK
acts as a PRR,binding to cytoplasmic DNA and stimulating the
expression of genesencoding type I IFNs (19). This study also
showed that Ku70 andSTING forma complex fromwhichKu70 dissociates 3
hours after stim-ulationwithDNA. Ferguson et al. (19) stated that
the dissociation ofKu70and STING activated downstream signaling and
induction of the ex-pression of genes encoding type I IFNs. Our
data are complementaryto these findings in demonstrating that
prolonged association of STINGandKu70may lead to the production of
type III IFN as a late event afterexposure toDNA.Here, we focused
on the late stage of the IFN immuneresponse.This time frame is
consistentwith the timecourseof IFN-l1pro-duction in response to
stimulation with DNA (fig. S1), which is alsoconsistent with our
previous study (31). The result from ELISA demon-strated that there
was a marked increase in the amount of IFN-l1 pro-tein secreted
into the cell culture medium 24 to 48 hours aftertransfection.
Furthermore, these experiments showed that detectableamounts of
IFN-a, IFN-b, and IFN-l2/3 were found in the cell culturemedium but
at a much lower abundance than that of IFN-l1 (fig. S1).Thus, the
Ku70-mediated production of IFN-l1 in response to expo-sure to DNA
or infection with a DNA virus is a dominant responsein HEK 293
cells, THP-1 cells, and primary human macrophages.
Ku70 is composed of fourmajor domains: thea/b domain,
theDNAbinding domain (b-barrel), the linker domain, and the
C-terminal do-main (41, 42). Here, we identified that the DNA
binding domain wasessential for the binding of Ku70 to STING. Ku70
and Ku80 form het-erodimers, and the absence of one subunit
destabilizes the other (43, 44).Both the Ku heterodimer (42) and
DNA-PKcs (45) can bind directly toDNA; however, in the absence of
either Ku70 or Ku80, the affinity ofDNA-PKcs for DNA is
substantially reduced (46). These findings sug-gest that each
component in the DNA-PK complex has an importantrole to
play.However, we further found thatKu80, evenwhenpresent inthe
Ku70-STING complex, was not functional in the DNA-mediatedinduction
of IFN-l1 production (fig. S5). DNA-PKcs was not necessar-ily
required for the interaction of the DNA binding domain of Ku70with
STING (fig. S5A), whereas this DNA binding domain itself
wasfunctional in the DNA-mediated induction of IFN-l1
production(fig. S5B). In summary, although Ku80 or DNA-PKcs was
present inthe complex containing Ku70 and STING, they were not
functionalin the induction of IFN-l1 production in response to
DNA.
Previous studies identified the STING-TBK1-IRF3 pathway as
play-ing an important role in the induction of type I IFN
production in re-sponse to different forms of nucleic acids (47,
48). Here, we providedfurther evidence to show that the
transcription factors IRF1, IRF7,and IRF3 were all involved in the
DNA-dependent induction of IFN-l1 production. A major distinction
between IRF3 on the one hand andIRF1 and IRF7 on the other is that
IRF3 is constitutively found in mostcell types, whereas IRF1 and
IRF7 are found in cells only after the ex-posure of cells to type I
IFNs (49). Our data confirmed that production
ββ-Actin
STING
A
B
0200400600800
Rel
ativ
e IF
NL
1 m
RN
A
abun
danc
e
0
400
800
1200
1600IF
N-λ
1, p
g/m
l
0
200
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800
Rel
ativ
e IF
NL
1 m
RN
A
abun
danc
e
0
400
800
1200
1600
IFN
-λ1,
pg/
ml
D
C
E
Fig. 7. Knocking down STING inhibits the production of IFN-l1by
humanMDMstransfected with DNA or infected with HSV-2. (A to E)
Human macrophages wereleft untreated, were mock-transduced, or were
transduced with lentivirus expressingcontrol shRNA (Lenti-shCtrl)
or shRNA targeting STING (Lenti-shSTING). (A) Seventy-twohours
after transduction, the indicated cells were analyzed by Western
blotting withantibody against STINGprotein.Western blots are
representative of three independentexperiments. (B and C) The
indicated transduced macrophages were transfected
withlinearizedDNA. Total cellular RNAwas then analyzed by real-time
RT-PCR to determinethe relative abundance of IFNL1mRNA (B), whereas
cell culture medium was analyzedby ELISA to determine the amounts
of IFN-l1 protein (C). Data aremeans ± SD of threeindependent
experiments. (D and E) The indicated transduced macrophages
wereinfected with HSV-2 at a multiplicity of infection (MOI) of
0.15. Total cellular RNA wasthen analyzedby real-timeRT-PCR
todetermine the relative abundance of IFNL1mRNA(D), whereas cell
culture medium was analyzed by ELISA to determine the amounts
ofIFN-l1 protein (E). Data are means ± SD of three independent
experiments.
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of the IRF1 and IRF7 proteins was induced in DNA-transfected
cellsand was inhibited by knockdown of IRF3, indicating that IRF1
andIRF7 production required IRF3. Our data further showed that
there wasa certain amount of IRF3 that accumulated in the nucleus,
but the in-tensity of the signal did not show differences between
DNA-stimulatedand unstimulated HEK 293 cells or STING KO cells.
This finding sug-gests that IRF3 might be activated at an early
stage but that IRF3 is notresponsible for the late-stage induction
of IFN-l1 production. Thegenes encoding IRF1 and IRF7 were induced
through the activationof IRF3 at an early stage and then
contributed to the abundant induc-tion of IFN-l1 production at a
later stage. This observation led us todivide the model of
Ku70-mediated IFN-l1 production into two dis-tinct steps. First,
the immediate or early response involved a very low(or
undetectable) amount of IFN produced through the activation ofIRF3.
Second, a low level of synthesis of IFNs from the first step
resultsin positive autocrine feedback by the production of IRF1 and
IRF7,which provides efficient amplification of IFN-l1 production at
a laterstage (49). This two-stage model is consistent with the time
course ofDNA-stimulated, Ku70-mediated IFNproduction (fig. S1). In
cells trans-fectedwithDNA,wewere unable to detect the expression of
IFNA, IFNB,or IFNL2/3 at earlier times (3 and 6 hours), and we
detected IFN-l1 atlowabundance.Robust amounts of IFN-l1were
produced at a later stage,that is, at 24 to 48 hours after the
cells were stimulated with DNA. Thisresult further suggested that
the production of IFN-a, IFN-b, and IFN-l2/3 was simultaneously
induced together with that of IFN-l1. The sameinduction kinetics
implied that the induction of production of these fourIFNs shared a
similar signaling transduction pathway in response to exog-enous
DNA, in which Ku70 and STING played an important role in
thedominant induction of IFN-l1 production with the simultaneous
induc-tion of IFN-a, IFN-b, and IFN-l2/3 production to a much
lesser extent.
In addition, this Ku70-STING-IRF3-, IRF1-, and
IRF7-mediatedpathway to induce IFN-l1 production differs from the
IFI16-STING-IRF3–mediated IFN-l1 pathway, which we have previously
reported(32), because IFI16-induced IFNL1 signaling peaked at 6
hours afterstimulationwithDNA stimulation, and IRF1 and IRF7 are
not involved(32). Note that the exogenous DNA that we used for the
induction ofIFN-l1 in the Ku70 pathway is distinct from theDNA that
was used forthe stimulation of the IFI16 pathway. In our current
study of the Ku70pathway in the induction of IFN-l1 production,
linearized DNA andHSV-2 were used, whereas in the study of the
IFI16-mediated pathwayfor the induction of IFN-l1 production,
circular DNA andHSV-1 viruswere used (15, 32). The knockdown of
IFI16 has no effect on the DNA-induced induction of IFN-l1,
indicating that IFI16 is not involved in theKu70-mediated IFN-l1
induction pathway (fig. S4). Therefore, it is stillan interesting
research area to discover the mechanism of distinct path-ogen
recognition patterns in host cells in response to very similar
for-eign invasion. Together, our results suggest that STING is an
essentialmediator downstream of Ku70 for the DNA-induced production
ofIFN-l1. This finding advances our understanding of the
regulationof the innate immune response and expands the current
repertoire ofDNA-sensing mechanisms. These findings may be of value
in further-ing our understanding of the function of the immune
system in healthand disease.
MATERIALS AND METHODSCells, antibodies, and virusesHEK 293
cells, 293T cells, and THP-1 cells were obtained from theAmerican
Type Culture Collection (ATCC) and maintained according
Sui et al., Sci. Signal. 10, eaah5054 (2017) 18 July 2017
to the manufacturer’s instructions. CD14+ monocytes were
purifiedfrom the peripheral blood mononuclear cells of healthy
donors usingCD14 MicroBeads (Miltenyi Biotec) according to the
manufacturer’sinstructions, as previously described (50). To
generate MDMs, isolatedCD14+monocyteswere plated on a 10-cmpetri
dish at 10 × 106 cells perdish. Monocytes were stimulated with
macrophage colony-stimulatingfactor (25 ng/ml) (R&D Systems) in
macrophage serum-free medium(Thermo Fisher Scientific) for 7 days.
MDMs were then maintained inDulbecco’s modified Eagle’s medium
(Thermo Fisher Scientific)containing 10% fetal bovine serum (FBS)
(HyClone Laboratories),25 mM Hepes (Quality Biology), and
gentamicin (5 mg/ml) (ThermoFisher Scientific) before they were
used in experiments. HSV-2 was ob-tained from Advanced
Biotechnologies Inc., and viral titers weredetermined by
plaque-forming assays with Vero cells (ATCC) (51).Antibodies used
in this study were as follows: anti-STING, anti-MyD88,
anti–b-catenin, anti-Ku80, anti-IRF1, anti-IRF7, and
anti-Flagantibodies were from Cell Signaling Technology; anti-Ku70
and anti–DNA-PKcs antibodies were from Abcam; and anti-IRF3
antibody wasfrom OriGene. Alexa Fluor 488– and Alexa Fluor
555–labeledsecondary antibodies were purchased from Cell Signaling
Technology.
Gel electrophoresis and Western blotting analysisCell lysates
were prepared with radioimmunoprecipitation assay buffer(Boston
BioProducts) in the presence of protease inhibitor
cocktail(Sigma-Aldrich) andHalt phosphatase inhibitor cocktail
(Thermo FisherScientific). Nuclear protein was extracted with the
Nuclear Extract Kit(Active Motif) according to the manufacturer’s
instructions. The proteinconcentrations of the cell lysates were
quantified with the bicinchoninicacid protein assay (Thermo Fisher
Scientific) to ensure that equalamounts of total protein were
loaded in each well of NuPAGE 4 to12% bis-tris gels (Thermo Fisher
Scientific). Proteins were transferredonto a nitrocellulose
membrane and analyzed byWestern blotting withthe appropriate
antibodies, which was followed by incubation withhorseradish
peroxidase–conjugated secondary antibodies and detectionof bands
with ECL plus Western blotting detection reagents (GEHealthcare).
Band intensities were analyzed with National Institutesof Health
Image J software (http://rsbweb.nih.gov/ij/).
PlasmidsThe PCR2.1 plasmid (Thermo Fisher Scientific) was
digested with EcoRI, and this digested plasmid was used as a
noncoding DNA stimulant.The Myc-tagged STING expression vector was
constructed as follows.STING-encoding complementary DNA (cDNA) was
synthesized fromthe total cellular RNA of HEK 293 cells using the
Superscript First-Strand Synthesis System for RT-PCR (Thermo Fisher
Scientific). Thereverse-transcribed cDNA encoding STING was
subcloned intopEF6/Myc-His (ThermoFisher Scientific). The
insertionwas confirmedby DNA sequencing with BigDye version 3.0
(Applied Biosystems).Other expression vectors used in this
studywere constructedwith a sim-ilar method. The cDNA encoding Ku70
was cloned into pEF1-Flag(Thermo Fisher Scientific). The DNA
encoding the full-length IFNL1promoter region [993 base pairs (bp)]
was amplified from HEK 293genomic DNA with the Expand high-fidelity
PCR system (RocheMolecular Biochemical) with a primer pair
(5′-GAGCTCAAAC-CAATGGCAGAAGCTCC-3′ and
5′-AGATCTTGGCTAAATCG-CAACTGCTTCC-3′). The 993-bp fragment was
subcloned andinserted into the vector pGL4.10 (Promega). The
presence of theintended fragment without any unexpected mutations
was confirmedby DNA sequencing, and this vector was named
pGL4–IFN-l1. The
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plasmid pTK-RL, which expresses Renilla luciferase under the
controlof HSV thymidine kinase promoter, was obtained from Promega
andused to normalize experimental variations.
IFN-l1 reporter assay293T cells (30 × 103 cells per well) were
seeded in six-well plates andcotransfected with 100 ng of
pGL4–IFN-l1, 10 ng of pTK-RL, and theappropriate amounts of STING
expression vector, wild-type Ku70 ex-pression vector, or mutant
Ku70 expression vector. The cells were thentransfected on day 2
with 1 mg of linearized DNA (as a stimulant), andthe cells were
then collected for the luciferase assay. The luciferase assaywas
performedwithDual-Glo luciferase assay system reagents
(Promega).Relative luciferase activity was calculated on the basis
of the ratio of theluminescence of firefly luciferase to that of
Renilla luciferase.
RNA extraction and real-time RT-PCRTotal cellular RNAwas
isolated from cells with the RNeasy isolation kit(Qiagen). The cDNA
was synthesized from total RNA with TaqManreverse transcription
reagents (Thermo Fisher Scientific) with randomhexamers as primers,
according to themanufacturer’s instructions. Therelative abundance
of IFNL1mRNAwasmeasured by quantitative RT-PCR on a CFX96 real-time
system (Bio-Rad); a two-temperature cycleof 95°C for 15 s and 60°C
for 1 min (repeated for 40 cycles) was used.Relative quantities of
IFNL1 transcripts were calculated with the DDCtmethod with
GAPDHmRNA as a reference. Normalized samples wereexpressed relative
to the averageDCt value for controls to obtain relativefold changes
in mRNA abundance.
Generation of STING KO cellsSTING KO cells were generated from
HEK 293 cells with the CRISPR/Cas9 genomic editing kit (OriGene)
according to the manufacturer’sinstructions. HEK 293 cells (5 ×
105) were transfected with 600 ng ofhCas9 target guide RNA and 600
ng of donor vector in a six-well tissueculture plate. The cells
were split at a 1:10 dilution 2 days after transfec-tion. After an
additional 3 days in culture, the cells were split again at a1:10
dilution. This procedure was repeated seven times. The cells
werethen transferred to 10-cm dishes and cultured withmedium
containingpuromycin (2 mg/ml) (Thermo Fisher Scientific). The
culture mediumwas changed every 2 to 3 days. Individual cell
colonies were isolated bylimiting dilution. After 1 to 2 weeks, the
cells were observed under themicroscope, and cells from those wells
containing only one cell colonywere selected and allowed to expand
from a 96-well to a 6-well plate.
Coimmunoprecipitation assaysHEK 293T cells (1 × 106) were seeded
onto a 100-mm dish and trans-fected with a Flag-tagged Ku70/Ku70
mutant expression vector and aMyc-tagged STING expression vector
using a TransIT-293 Transfec-tion Kit (Mirus Bio LLC). The cells
were further transfected on day 2with 10 mg of linearized DNA (as a
stimulant). The cells were lysed inPierce IP lysis buffer (Thermo
Fisher Scientific) with 10% glycerol(Sigma-Aldrich). The lysates
were centrifuged at 10,000g for 10 minat 4°C to remove cell debris.
The supernatants were immunoprecipi-tated with an anti-Flag agarose
(Sigma-Aldrich) or anti–c-Myc agarose(Thermo Fisher Scientific) and
then analyzed byWestern blotting withanti-FLAG or anti-STING
antibodies.
Confocal microscopyCells grown on 35-mm glass bottom dishes were
fixed in 4% para-formaldehyde, blocked in blocking buffer [1×
phosphate-buffered sa-
Sui et al., Sci. Signal. 10, eaah5054 (2017) 18 July 2017
line, 5% normal goat serum (Cell Signaling), and 0.1% saponin
(AlfaAesar)] for 1 hour, stained overnight with primary antibodies
(dilutedat 1:200) at 4°C, and then incubated with Alexa Fluor 488–
or AlexaFluor 555–labeled secondary antibodies (1:1000) for 1 hour.
Bottom-coated coverslips were mounted with ProLong Diamond
AntifadeMountant withDAPI (Thermo Fisher Scientific). Imageswere
capturedon an LSM 710 scanning confocal microscope.
Lentivirus packaging and transductionA lentiviral plasmid
containing an expression cassette encodingSTING-specific shRNA was
purchased from OriGene. Lentiviral parti-cles were packaged in 293T
cells according to the manufacturer’s in-structions. Harvested
lentivirus-containing cell culture medium wasconcentrated with a
lenti concentrator (OriGene). The lentiviral parti-cleswere
titrated on the basis of their abundance of theHIVp24
antigenmeasured with an HIV-1 p24 ELISA kit (PerkinElmer). For
lentiviraltransductions, human macrophages were seeded on six-well
plates to70% confluence and inoculated with lentiviruses at an MOI
of 50in culture medium containing 2% FBS in the presence of
polybrene(8 mg/ml). Twenty-four hours after the cells were
transduced, the me-dium was replaced by fresh complete culture
medium, and the cellswere then incubated for a further 72 hours to
enable protein knockdownto occur.
Enzyme-linked immunosorbent assayThe amounts of IFN-a, IFN-b,
IFN-l1, and IFN-l2/3 proteins in cell cul-ture medium were measured
with the VeriKine-HS Human IFN-a AllSubtype ELISA Kit (PBL Assay
Science), the VeriKine-HS HumanIFN-b Serum ELISA Kit (PBL Assay
Science), the human IL-29/IFN-l1DuoSet ELISA (R&D Systems), and
the DIY Human IFN-l2/3 (IL-28A/B)ELISA (PBL Assay Science) kits,
respectively, according to the manufac-turers’ protocols. The
minimum detectable concentrations of IFN-a,IFN-b, IFN-l1,
andIFN-l2/3were1.95,1.2,62.5, and125pg/ml, respectively.
Statistical analysisResults were representative of at least
three independent experiments.The values are expressed as means ±
SD of individual samples. Statis-tical significance was determined
by Student’s t test. P < 0.05 wasconsidered to indicate a
statistically significant difference between theexperimental
groups.
SUPPLEMENTARY
MATERIALSwww.sciencesignaling.org/cgi/content/full/10/488/eaah5054/DC1Fig.
S1. Time course of theDNA-stimulated inductionof IFN-a, IFN-b,
IFN-l1, and IFN-l2/3 production.Fig. S2. Comparison of the
endogenous amounts of b-catenin and MyD88 proteins betweenHEK 293
cells and 293T cells.Fig. S3. Analysis of the production of IFN-a,
IFN-b, and IFN-l2/3 in THP-1 cells transfected with DNA.Fig. S4.
Knockdown of IFI16 has no effect on the Ku70-mediated induction of
IFN-l1production in response to exogenous DNA.Fig. S5. Ku80 and
DNA-PKcs are not involved in the Ku70-mediated induction of
IFN-l1production in response to exogenous DNA.Fig. S6. The
activation of IRF1, IRF3, and IRF7 involves the Ku70-mediated
induction of IFNproduction in response to exogenous DNA.Fig. S7.
Knockdown of STING inhibits the production of IFN-a, IFN-b, and
IFN-l2/3 by humanMDMs transfected with DNA or infected with
HSV-2.
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Acknowledgments: We thank H. Young for discussion, S. Lockett
and K. Peifley for technicalsupport on the confocal microscope, and
Q. Chen for assistance with supplies. Funding:This project was
funded in whole or in part with federal funds from the National
Cancer Institute,NIH under contract no. HHSN261200800001E. The
content of this publication does not necessarilyreflect the reviews
or policies of the U.S. Department of Health and Human Services nor
doesmention of trade names, commercial products, or organizations
imply endorsement by theU.S. government. Author contributions: H.S.
designed and performed experiments; H.S., H.C.L.,and T.I. discussed
and interpreted the data; T.I. and H.I. supported constructing the
Ku70 mutants;X.J. and B.T.S. analyzed the confocal images; M.Z.
performed mass spectrometry analysis; andH.S., H.C.L., and T.I.
wrote the manuscript. Competing interests: The authors declare that
theyhave no competing interests.
Submitted 6 July 2016Resubmitted 27 September 2016Accepted 30
June 2017Published 18 July 201710.1126/scisignal.aah5054
Citation: H. Sui, M. Zhou, H. Imamichi, X. Jiao, B. T. Sherman,
H. C. Lane, T. Imamichi, STING isan essential mediator of the
Ku70-mediated production of IFN-l1 in response to exogenousDNA.
Sci. Signal. 10, eaah5054 (2017).
11 of 11
http://stke.sciencemag.org/
-
exogenous DNA1 in response toλSTING is an essential mediator of
the Ku70-mediated production of IFN-
Hongyan Sui, Ming Zhou, Hiromi Imamichi, Xiaoli Jiao, Brad T.
Sherman, H. Clifford Lane and Tomozumi Imamichi
DOI: 10.1126/scisignal.aah5054 (488), eaah5054.10Sci.
Signal.
phase of the antiviral IFN response to DNA viruses.type III IFN
production was slower than that of type I IFN. Together, these data
suggest a role for STING in the late mediator of type I IFN
production. However, in the Ku70 pathway, IRF1 and IRF7 were
required in addition to IRF3, andKu70 translocated from the nucleus
to the cytosol to interact with the adaptor protein STING, a
well-characterized
1 in human cells exposed to cytosolic DNA or infected with the
DNA virus HSV-2.λproduction of the type III IFN
IFN-inducedinvestigated the mechanism by which the DNA protein
kinase (DNA-PK) component Ku70, a DNA repair protein,
et al.transcription factor IFN regulatory factor 3 (IRF3) to
induce expression of genes encoding type I IFNs. Sui part of the
antiviral response. Various cytosolic DNA sensors and their
downstream effectors stimulate activation of the
When cells sense cytosolic DNA, such as occurs during viral
infection, they produce type I interferons (IFNs) as1λSTINGing
viruses with IFN-
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