Rossana Trotta,1Li Chen,1David Ciarlariello,1Srirama Josyula,1Charlene Mao,1Stefan Costinean,2,3Lianbo Yu,4
Jonathan P. Butchar,2,5Susheela Tridandapani,2,5Carlo M. Croce,1,2and MichaelA. Caligiuri1,2,5,6
1Department of Molecular Virology, Immunology and Medical Genetics,2The Comprehensive Cancer Center,3Department of Pathology,4Center for Biostatistics,
5The Division of Hematology/Oncology, Department of Internal Medicine, and6The James Cancer Hospital and Solove Research Institute, The Ohio State
University, Columbus, OH
MicroRNAs (miRs) are small, noncoding
RNA molecules with important regulatory
functions whose role in regulating natural
killer (NK) cell biology is not well defined.
Here, we show that miR-155 is synergisti-
cally induced in primary human NK cells
after costimulation with IL-12 and IL-18,
or with IL-12 and CD16 clustering. Over-
expression of miR-155 enhanced induc-
tion of IFN-? by IL-12 and IL-18 or CD16
stimulation, whereas knockdown of miR-
155 or its disruption suppressed IFN-?
lated NK cells. These effects on the regu-
lation of NK cell IFN-? expression were
found to be mediated at least in part via
miR-155’s direct effects on the inositol
phosphatase SHIP1. Consistent with this,
we observed that modulation of miR-155
overrides IL-12 and IL-18–mediated regu-
lation of SHIP1 expression in NK cells.
Collectively, our data indicate that miR-
155 expression is regulated by stimuli
that strongly induce IFN-? in NK cells
such as IL-12, IL-18, and CD16 activation,
and that miR-155 functions as a positive
regulator of IFN-? production in human
NK cells, at least in part via down-
regulating SHIP1. These findings may
have clinical relevance for targeting miR-
155 in neoplastic disease. (Blood. 2012;
Human natural killer (NK) cells are CD56?CD3?large granular
lymphocytes of the innate immune system.1,2NK cells participate
in early responses against infection or malignant transformation. In
addition to their potent cytolytic activity, NK cells have an
important immunoregulatory function in that they produce cyto-
kines and chemokines when activated. In particular, NK cells
produce IFN-?, a critical cytokine for the clearance of infectious
pathogens and tumor surveillance,3in response to a wide variety of
stimuli, including both soluble factors and cellular interactions.4,5
Dendritic cells and monocytes stimulated with bacterial cell wall
components release monokines such as IL-12 and IL-18, which
synergistically induce rapid and robust production of IFN-? by
NK cells also express the low-affinity receptor for the Fc
fragment of immunoglobulin (Ig)G (Fc?RIIIA, CD16), which is
the activating receptor required for triggering antibody dependent
cellular cytotoxicity (ADCC) as well as the induction of IFN-?.7
IL-12 monokine stimulation in combination with CD16 activation
induces a synergistic induction of IFN-? in NK cells, but to a lesser
extent than does IL-12 and IL-18 costimulation.8This observation
has recently been shown to have implications in the antibody
therapy of breast cancer patients. In fact, the antitumor actions of
the anti-HER2 monoclonal antibody trastuzumab are enhanced by
IL-12 treatment in vivo, and this effect is dependent on NK cell
production of IFN-?.9
The regulation of NK cell IFN-? production involves positive
and negative mediators, such as kinases and phosphatases, as well
as transcription factors.10-14SHIP1 is a hematopoietic cell specific
5? inositol phosphatase.15We have previously shown that SHIP1 is
expressed differentially in CD56brightand CD56dimNK cell subsets,
and is negatively modulated by the costimulation of IL-12 and
IL-18.13SHIP1, by dampening the PI3K pathway, is able to
negatively regulate IFN-? production by monokines and CD16
stimulation in both human and mouse NK cells.13,16
MicroRNAs (miRs) are a highly conserved class of small,
noncoding RNAs with important regulatory functions in prolifera-
tion, differentiation, signal transduction, immune responses, and
carcinogenesis.17miRs regulate gene expression posttranscription-
ally by forming imperfect base pairs with sequences in the
3? untranslated region (UTR) of genes. In turn, this prevents protein
accumulation by repressing translation or by inducing mRNA
degradation.18Recently, a general role of miRs in regulation of NK
cell activation, survival, and function has been shown using
conditional deletion of Dicer or Dgcr8.19Aspecific role of miR-150
in regulating development and maturation of mouse NK cells has
also been reported.20Further, it has been shown that miR-181
promotes human NK cell development by regulating Notch signal-
ing.21In addition, Fehninger et al have shown that treatment of
mouse NK cells with IL-15 increased or decreased the expression
of several miRs.22Among these miRs, miR-223 was down-
modulated thereby up-regulating its target gene, granzyme B.22
Among miRs, miR-155 is involved in protective immunity when
properly regulated and contributes to malignant transformation
when deregulated.23miR-155 is processed from the non–protein-
coding transcript of the BIC gene RNA. Further, it is required for
the normal function of B, T, and dendritic cells,24,25and its
expression is increased during B, T, macrophage and dendritic cell
activation.23Transgenic mice with selective overexpression of
Submitted December 16, 2011; accepted February 13, 2012. Prepublished
online as Blood First Edition paper, February 29, 2012; DOI 10.1182/blood-
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
© 2012 by TheAmerican Society of Hematology
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miR-155 in B cells develop B-cell lymphoma.26Of interest,
miR-155 is overexpressed in NK-cell lymphoma/leukemia and this
correlates with low levels of SHIP1 expression and up-regulation
of AKT signaling.27Still, the expression and role of miR-155 in
regulating NK cell development and function have yet to be
explored. In this report, we characterize the expression of miR-155
in human NK cells, its role in the regulation of NK cell IFN-?
expression, and the mechanism by which this occurs.
Cells lines, NK cell preparations, and flow cytometry
The human IL-2 dependent NK cell line NK-92 (gift of Dr H. Klingemann,
Rush Cancer Center, Chicago, IL) was maintained in culture in RPMI-1640
medium (Invitrogen), supplemented with 20% heat-inactivated FBS (Invit-
rogen), 2mM L-glutamine and 150 IU/mL rhIL-2 (Hoffman-LaRoche). The
amphotropic-packaging cell line 293T was maintained in culture in
Dulbecco modified Eagle medium (DMEM; Invitrogen)/10% FBS medium
and grown for 16 to 18 hours to 80% confluence before transfection by
calcium phosphate-DNA precipitation (Profection System; Promega).
Human NK cells were isolated from peripheral blood leukopacks of healthy
individuals (American Red Cross) by incubation for 30 minutes with
RosetteSep NK cell antibody cocktail (StemCell Technology), followed by
Ficoll-Hypaque density gradient centrifugation. NK cell preparations
containing ? 99% CD56?NK cells were obtained by positive selection
using CD56 MicroBeads and MACS separation columns from Miltenyi
(Miltenyi Biotech), as determined by direct immunofluorescence using an
anti-CD56 phycoerythrin (PE)–conjugated monoclonal antibody (Ab; Im-
munotech). IL-18R? fluorescence-activated cell sorter (FACS) analysis
was performed by staining CD56?NK cells with IL-18R? PE-conjugated
Ab or PE-conjugated nonreactive isotype control Ab (R&D Systems).
Samples were acquired using FACSCalibur or LSRII (BD Bioscience) and
analyzed with FlowJo Version 7.6.1 (TreeStar). Values for both percent
positivity and mean fluorescence intensity (MFI) were obtained and
recorded.All work with human materials was approved by the institutional
review board of The Ohio State University Comprehensive Cancer Center.
Wild-type (WT; Bic?/?) and miR-155?/?(Bic?/?) mice (B6, 129S7-
Mirn155tm/Brd) were obtained from Mutant Mouse Regional Resource
Centers.28In the spleens of miR-155 KO compared withWTlittermates, we
observed a similar percentage and total number of NK.1?CD3?NK cells
and similar levels of CD94 surface expression (data not shown).All animal
work was approved by The Ohio State University Animal Care and Use
Committee, and mice were treated in accordance with the institutional
guidelines for animal care.
Lentivirus infection of NK-92 cell line and primary human NK
Lentiviral vectors encoding miR-155 (Lenti-miR-155), the antisense (miR-
Zip155), and empty vectors (Lenti and miR-Zip00) were obtained from SBI
System Biosciences. NK-92 cells and primary NK cells were infected
followed protocol similar to previously published standards.29Briefly,
infectious supernatant from Lenti, Lenti-miR-155, miR-Zip00, and miR-
Zip155 transfected 293T cells were collected after 48 hours and used for
2 or 3 cycles of infections at multiplicity of infection of 10. All vectors
contain the gene for green fluorescent protein (GFP). On infection, NK-92
cells and CD56?NK cells were sorted for GFP expression on a FACSAria
II (BD Bioscience). GFP?CD56?primary NK cells were used for
experimentation immediately after sorting. Expression of miR-155 was
confirmed by real time RT-PCR (reverse transcription [transcriptase]-
polymerase chain reaction) in NK-92 cells.
Cell culture conditions
Before cytokine stimulation, NK-92 cells were cultured in IL-2–free
medium containing 10% FBS for 18 or 36 hours. NK-92 and primary
NK cells were incubated in medium plus 10% or 20% FBS at 37°C
(1 ? 106/mL) for the indicated times with the addition of recombinant
human or mouse IL-12 (kindly provided by Genetics Institute) or recombi-
nant human or mouse IL-18 (R&D Systems) at the indicated concentration.
For experiments using immobilized Abs, wells of flat bottom plate were
coated with PBS diluted IgG (100 ?g/mL; Sigma-Aldrich) or anti–mouse
CD16 (10 ?g/mL; R&D Systems) overnight at 4°C.
Western blot analysis
RIPA buffer: 0.15M NaCl, 1% NP-40, 0.1% SDS, 50mM Tris, pH 8.0),
supplemented with protease and phosphatase inhibitors, 1mM phenylmeth-
ylsulfonylfluoride (PMSF), 1mM Na3VO4, 50mM NaF, 10mM ?-glycerol-
phosphate, 1mM ethylenediaminetetraacetic acid (EDTA), and a protease
inhibitor cocktail tablet from Roche Applied Science, as described.30
Alternatively, cells were directly lysed in Laemmli buffer (2 ? 105
cells/20 ?L). Western blotting was performed according to previously
ish peroxidase–labeled sheep anti–rabbit, mouse and/or goat Ig sera and
enhanced chemiluminescence (ECL; Amersham). Proteins were analyzed
in 4%-15% sodium dodecyl sulfate (SDS)–polyacrylamide gel electropho-
resis (PAGE; Bio-Rad Laboratories) using reducing conditions. To measure
the levels of protein expression, the intensity of each band was quantified by
densitometry using ImageJ Version 1.43 software (National Institutes of
Health; http://rsb.info.nih.gov/ij/docs/index.html), and normalized to the
level ofACTIN or GRB2 in each lane. Monoclonal and polyclonalAbs used
were: mouse monoclonal anti-SHIP1 and the goat anti-ACTIN from Santa
Cruz Biotechnology; and monoclonal anti-GRB2 Ab from Transduction
Total RNA was extracted using either RNeasy Mini kits (QIAGEN) or
Trizol (Invitrogen). cDNA was generated according to the manufacturer’s
recommendations (Invitrogen) or TaqMan MicroRNAreverse transcription
kit and RT primers specific for miR-155 and RNU44 as control (Applied
Biosystem). Real-time RT-PCR reactions were performed as a reaction with
primer/probe set specific for the human BIC, SHIP1, miR-155, RNU44
(Applied Biosystem), IFN-?, and 18S as previously described.12Water (no
template) was used as a negative control. Reactions were performed using
an ABI prism 7700 sequence detector (Taqman; PE Applied Biosystems),
and data were analyzed with the Sequence Detector Version 1.6 software to
establish the PCR cycle at which the fluorescence exceeded a set threshold,
CT, for each sample. Data were analyzed according to the comparative
CTmethod using the internal control RNU44 and 18S transcript levels to
normalize differences in sample loading and preparation. Results represent
the n-fold difference of transcript levels in a particular sample compared
with calibrator cDNA samples of unstimulated or control vector infected
NK-92 or primary NK cells. Results are expressed as the mean ? SEM of
triplicate reaction wells.
IFN-? ELISA assays
Quantification of human IFN-? was performed using commercially avail-
able mAbs pairs (Endogen). Cell free supernatants were collected after
18 or 24 hours of incubation at 37°C with the indicated stimuli. WT and
miR-155?/?purified mouse NK1.1?CD3?NK cells were left untreated or
stimulated with monokines and/or immobilized Abs for 18 or 24 hours at
37°C. Cell supernatants were collected and analyzed by enzyme-linked
immunosorbent assay (ELISA) using a Kit from R&D Systems. Results are
shown as the mean of triplicate wells ? SEM.
Data were compared using student 2-tailed t test. A P value ? .05 was
miR-155 IN NK CELLS3479BLOOD, 12APRIL 2012?VOLUME 119, NUMBER 15
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Expression of miR-155 and BIC in resting and
monokine-activated human NK cells
Stimulation with the combination of IL-12 and IL-18 cytokines
induces a rapid and high induction of IFN-? expression in
NK cells.6An Affymetrix profile identified miR-155 as the most
highly induced miR by human NK cell costimulation with IL-12
and IL-18 cytokines (data not shown), which was further quantified
by performing real-time RT-PCR in 7 different fresh primary
human NK samples.After culture in IL-12 plus IL-18 we observed
an average induction in miR-155 expression of 27.8-fold
(P ? .0001; Figure 1A), but only 3.7-fold with IL-18 alone
(P ? .01, n ? 4) and zero with IL-12 alone. In further support of
these data, the non–protein-coding miR-155 precursor RNA BIC
this induction with IL-12 plus IL-18 were similar to those of IFN-?
release (Figure 1C).6Furthermore, activated CD56brightNK cells show
yet express low levels of miR-155 compared with CD56dimNK cells in
both resting (P?.001; Figure 1D), and IL-12 and IL-18 costimulated
To better understand the basis for this synergistic induction of
miR-155 in the presence of IL-18 and IL-12, we assessed IL-18
receptor (R) surface density expression in cytokine stimulated
human NK cells. Stimulation of resting NK cells with IL-12 alone
significantly induced IL-18R?, whereas costimulation with IL-12
plus IL-18 induced significantly more IL-18R? expression as
detected by percent of expression and mean fluorescence intensity
(Figure 2A-B). Conversely, IL-18R? was not visibly induced by
IL-12 stimulation alone; however, the combination of IL-12 plus
IL-18 led to strong induction (data not shown).
To determine whether the synergistic induction of miR-155 is
dependent on the induction of IL-18R, we stimulated primary
CD56?NK cells overnight with the combination of IL-12 and
IL-18 and sorted cells into 2 groups: high expression of IL-18R?
and low expression of IL-18R?. These sorted populations were
then analyzed for miR-155 and IFN-? mRNA (Figure 2C-D). We
observed that expression of IL-18R? directly and significantly
correlated with levels of miR-155 (P ? .001; Figure 2C) and IFN-?
The role of miR-155 in the regulation of IFN-? production by
IL-12 plus IL-18–stimulated NK cells
Based on our observation that both miR-155 and IFN-? are
synergistically induced by IL-12 plus IL-18 in human NK cells, we
hypothesized that miR-155 could be a positive regulator of IFN-?
production. Thus, we overexpressed miR-155 by infecting the
CD3?CD56?human NK cell line NK-92 and CD3?CD56?
primary human NK cells using a lentiviral vector carrying either
the cDNA for GFP alone (Lenti), or GFP plus the miR-155 native
stem-loop structure along with 200 to 400 base pairs of upstream
and downstream flanking genomic sequence (Lenti-miR-155).
Overexpression of miR-155 was confirmed in the GFP?fraction
after NK-92 cell sorting (Figure 3A). Importantly, overexpression
of miR-155 led to a significantly greater production of IFN-? in
GFP?primary human NK cells after costimulation with IL-12 plus
IL-18, compared with the mock-infected primary human NK cells
(*P ? .01; Figure 3B).
To confirm our observation, we down-modulated miR-155 by
infecting NK-92 cells using an empty lentiviral vector called
miR-Zip00, or a lentiviral vector encoding antisense miR-155
called miR-Zip155. In the human NK-92 cell line, down modula-
tion of miR-155 significantly lowered IFN-? production at both
protein (*P ? .01, n ? 3) and mRNA (*P ? .001, n ? 3) levels
after stimulation by IL-12 plus IL-18 compared with mock-infected
NK-92 cells (Figure 4A). To address this observation in primary
NK cells, we turned to Bic?/?mice. BIC is the precursor mRNAto
miR-155, and Bic?/?and Bic?/?mice were therefore used in this
experiment. Primary NK cells from Bic?/?mice also showed
significantly less IFN-? production after stimulation with IL-12
and IL-18, compared with NK cells from Bic?/?mice (*P ? .001,
n ? 3; Figure 4B).
Regulation of SHIP1 expression in resting and IL-12 and/or
IL-18 activated primary human NK cells
SHIP1 5? inositol phosphatase is a negative regulator of IFN-? in
both human and mouse NK cells,13,16and a primary target of
miR-155.28,32,33To address whether there is an inverse correlation
between SHIP1 expression and miR-155 expression in NK cells,
we first analyzed SHIP1 mRNA and protein expression in primary
NK cells stimulated with IL-12 and/or IL-18. SHIP1 was expressed
by the stimulation with IL-18 alone and substantially reduced
further by the combination of IL-12 plus IL-18 stimulation, but no
change was seen with IL-12 alone (Figure 5A-C).
Monokine-mediated regulation of SHIP1 expression in human
NK cells is mediated via miR-155
To determine whether miR-155 regulates SHIP1 expression in
human NK cells, we first analyzed SHIP1 expression in NK-92
cells overexpressing sense or antisense miR-155, as previously
described. SHIP1 expression was down-modulated in NK-92 cells
overexpressing miR-155 (Figure 6A left) and up-regulated in cells
with reduced expression of miR-155 (Figure 6Aright). Next, in the
Figure 1. miR-155 and BIC RNA expression in resting and IL-12 and/or IL-18–stimulated NK cells and NK subsets. CD56?human NK cells were left unstimulated
or stimulated with IL-12 (10 ng/mL), IL-18 (100 ng/mL) or combination of both for 18 hours after which pellets were collected and used to prepare RNA. miR-155 (A) and
BIC (B) RNA expression were quantified by real-time RT-PCR. (C) Supernatants were analyzed for IFN-? production by ELISA. (D) CD56brightand CD56dimNK cells were
quantified for miR-155 expression by real-time RT-PCR. This experiment is representative of at least 4 such experiments performed with similar results.
3480 TROTTAet alBLOOD, 12APRIL 2012?VOLUME 119, NUMBER 15
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same cell lines, we analyzed the expression of SHIP1 after
costimulation with IL-12 plus IL-18. Ectopic expression of miR-
155 antisense rescued SHIP1 down-modulation (Figure 6B left
panel), whereas ectopic overexpression of miR-155 sense induced
additional down-modulation of SHIP1 (Figure 6B right panel).
These data provide evidence that miR-155 regulates SHIP1 in both
resting and cytokine-activated NK cells and that down-modulation
of SHIP1 in NK cells costimulated with IL-12 plus IL-18 is at least
partially regulated by and dependent on the expression of miR-155.
miR-155 expression and role in NK cells activated via CD16 and
We have previously shown that SHIP1 regulates IFN-? production
induced by FcR?IIIA (CD16) stimulation in both human and
mouse NK cells.16We therefore quantified miR-155 expression in
NK cells after activation via CD16 and/or IL-12, because in
combination, these signals result in a synergistic induction of
IFN-? production (Figure 1C).8Both miR-155 and its precursor
BIC were induced in primary human NK cells by CD16 stimulation
(P ? .01, n ? 4), whereas a greater induction of miR-155 and BIC
resulted from costimulation by CD16 and IL-12 (Figure 7A-B;
P ? .01, n ? 4). CD16 stimulation alone was able to significantly
reduce SHIP1 mRNA and protein expression in primary NK cells,
whereas costimulation via CD16 and IL-12 resulted in only a
modest yet significant further reduction of SHIP1 mRNA(P ? .01,
n ? 4) but not protein (Figure 7D-E).
The role of miR-155 in regulating IFN-? through CD16 and
IL-12 stimulation was analyzed using primary human NK cells
Figure 2. miR-155 expression in IL-18R?highand
IL-18R?lowNK cells stimulated by IL-12 plus IL-18.
(A) Freshly isolated CD56?human NK cells were stimu-
lated with IL-12 (10 ng/mL), IL-18 (100 ng/mL) or their
combination for 48 hours after which cells were analyzed
by FACS for expression of IL-18R?. A nonreactive di-
rectly conjugated isotype-identical Ab was used as a
control. (B) Percent and MFI of IL-18R? expression from
6 different donors are summarized. (C) NK cells activated
for 24 hours with IL-12 plus IL-18 were FACS sorted for
IL18R?highand IL18R?lowexpression, and quantified for
miR-155 and (D) IFN-? mRNA expression by real-time
RT-PCR. This experiment is representative of at least
4 experiments performed with similar results.
Figure 3. miR-155 overexpression enhances IL-12 plus IL-18–induced IFN-? production in NK cells. (A) NK-92 cells were infected using an empty lentivirus vector (Lenti)
containing GFP or a lentivirus vector containing GFP and miR-155 sense (Lenti-miR-155 vector). Cells were then sorted for high GFP expression and analyzed for miR-155
expression by RT-PCR. (B) Primary human CD56?NK cells were infected using either Lenti or Lenti-miR-155, sorted by FACS for GFP (left), plated in medium and
costimulated with IL-12 (10 ng/mL) plus IL-18 (100 ng/mL) or 1/5thor 1/25ththe doses of these monokines for 24 hours. Supernatants were then collected and assayed for
IFN-? by ELISA (right). These data are representative of 3 experiments performed with moderate doses, and 2 experiments performed with high and low concentrations of
miR-155 IN NK CELLS 3481 BLOOD, 12APRIL 2012?VOLUME 119, NUMBER 15
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overexpressing miR-155 and in primary NK cells from Bic?/?
mice. In primary human NK cells overexpressing miR-155, we
observed an increase in IFN-? after costimulation with CD16 and
IL-12 compared with mock-infected cells (Figure 7F). In contrast,
primary NK cells from Bic?/?mice showed a significant decrease
in IFN-? production after stimulation by CD16 and/or IL-12
compared with WT controls (*P ? .01, n ? 3; **P ? .04, n ? 3;
***P ? .001, n ? 3; Figure 7G).
IFN-? is the prototypic cytokine produced by NK cells. Indeed, its
fundamental role during inflammation and tumor immunity has
been well established, in that IFN-? deficiency results in increased
susceptibility to infection and/or malignancy.34,35In contrast,
overproduction of IFN-? can lead to autoimmune disorders.36
MicroRNAs have recently been shown to play a critical role in
the immune system as regulators of gene expression.37The
expression of miR-155 in NK cells and its role in regulating NK
cell functions has not been investigated. Here we show that
miR-155 functions as a regulator of IFN-? production in activated
human NK cells at least in part via the modulation of SHIP1
Among combinations of cytokines, IL-12 plus IL-18 potently
induces NK-cell IFN-? production, in part because of the synergis-
tic effect of these 2 monokines on this process.6In this report, we
provide clear evidence that like IFN-? production, the expression
of miR-155 and its precursor BIC are synergistically induced in
primary human NK cells after stimulation with IL-12 plus IL-18. In
Figure 4. Effect of miR-155 down-modulation on IFN-? production
in monokine-stimulated NK cells. (A) NK-92 cells were infected with
empty vector (miR-Zip00) or a miR-155 antisense encoding vector
(miR-Zip155), sorted for GFP?and analyzed for miR-155 expression by
real-time RT-PCR (left). GFP?miR-Zip00 and miR-Zip155 NK-92 cells
were stimulated with IL-12 (10 ng/mL) plus IL-18 (100 ng/mL) for 24 hours
and analyzed for IFN-? production by ELISA(middle) and real-time RT-PCR
(right). (B) BIC is the precursor of miR-155. NK cells sorted from spleens
of Bic?/?or Bic?/?mice were analyzed for miR-155 expression by
real-time RT-PCR (left), and stimulated with IL-12 (20 ng/mL) and IL-18
(10 ng/mL). Supernatants were then collected and analyzed for IFN-? by
ELISA (right). This experiment is representative of 3 performed with
Figure 5. Quantification of SHIP1 expression in monokine-stimulated primary human CD56?NK cells. (A) CD56?primary human NK cells were first incubated for
18 hours with medium containing either IL-12 (10 ng/mL) and/or IL-18 (100 ng/mL), and then quantified for SHIP1 (A) mRNAby real-time RT-PCR and (B) protein by Western
blot.The relative levels of SHIP1 protein, as determined by densitometry, are indicated above the blot and expressed as densitometric units relative to the control lane (“none”),
which is arbitrarily set at 1.00. (C) The graph summarizes the data of densitometric analysis of 3 primary donors. This experiment is representative of at least 3 such
experiments performed with similar results.
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fact, IL-18 stimulation alone but not IL-12 alone, modestly induced
miR-155 and BIC in human NK cells. Others have previously
shown that IL-12 treatment of PBMC induced IL-18R expression
in CD56?NK cells.38Here we observed that IL-12 specifically and
directly induced IL-18R? expression, but not IL-18R? expression
(not shown), on purified human NK cells. Further, we found a
direct correlation between IL-18R?, miR-155 and IFN-? expres-
sion in NK cells stimulated by IL-12 plus IL-18. These observa-
tions support a model where the synergistic induction of miR-155
by IL-12 and IL-18 depends at least in part on the induction of
IL-18R? by IL-12, and suggest that miR-155 is one of the
molecules responsible for the synergistic induction of IFN-? in
NK cells stimulated with IL-12 plus IL-18.
A characteristic of IL-18R signaling is the phosphorylation of
IL-1R-associated kinase (IRAK) and the sequential activation of
nuclear factor-?B (NF-?B).39,40NF-?B directly binds to the IFN-?
promoter,39but its activity is also directly correlated with miR-155
expression.41Our data suggest that induction of miR-155 by IL-18
in NK cells is probably mediated via NF-?B activation.
Importantly, by performing loss or gain-of-function experi-
ments in primary human and mouse NK cells and in the human
NK-92 tumor cell line, we identify miR-155 as a positive regulator
of IFN-? in NK cells.
Notably, despite having multiple targets that include MAF,
PU1, SOCS1, and Taff,23miR-155 appears to be the only known
miR that targets the 3?UTR of SHIP1 and regulates its expres-
sion.28,32,33Our previous study identified SHIP1 as a negative
regulator of IFN-?, with a higher level of constitutive expression in
resting CD56dimNK cells compared with CD56brightNK cells. One
would have therefore predicted that miR-155 expression would be
Figure 7. Expression of miR-155 and SHIP1 in CD16-activated NK cells, and the role of miR-155 in regulating IFN-?. (A) Primary human NK cells were activated with
immobilized human IgG and/or IL-12 (10 ng/mL) for 24 hours and quantified for miR-155 and (B) BIC mRNAexpression by real-time RT-PCR. (C) Supernatants were analyzed
for IFN-? production by ELISA. (D) SHIP1 mRNA expression was quantified by real-time RT-PCR and (E) protein expression by Western blot. (F) CD56?primary human NK
cells were infected using Lenti or Lenti–miR-155 vectors, sorted for GFP, and left unstimulated or stimulated with immobilized human IgG and/or IL-12 (10 ng/mL) for IFN-?
secretion. This experiment is representative of 2 experiments performed with identical results. (G) NK cells were isolated from spleens of Bic?/?and Bic?/?mice, cultured
8 days in medium containing IL-2 (900 IU/mL), stimulated for 24 hours in vitro with immobilized anti-CD16Ab and/or IL-12 (20 ng/mL) and assayed for IFN-?.
Figure 6. SHIP1 is regulated by miR-155 in resting and monokine-stimulated human NK cells. (A) GFP?NK-92 Lenti and GFP?NK92 Lenti-miR-155 cells (left) and
GFP?NK-92 miR-Zip00 and NK-92 miR-Zip155 cells (right) were cultured in medium without IL-2 for 24 hours and then quantified for SHIP1 protein expression by Western
blot. (B) Comparable NK-92 cells were then cultured in medium containing IL-12 (10 ng/mL) plus IL-18 (100 ng/mL) for 24 hours and then quantified for SHIP1 protein
expression by Western blot. The relative levels of SHIP1 protein, as determined by densitometry, are indicated above the blot and expressed as densitometric units relative to
the control lane (far left), which is arbitrarily set at 1.00. This experiment is representative of at least 2 such experiments performed with similar results.
miR-155 IN NK CELLS3483 BLOOD, 12APRIL 2012?VOLUME 119, NUMBER 15
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inversely correlated with SHIP1 expression in these resting NK
subsets; however, we found the opposite: CD56dimNK cells have a
higher level of miR-155 expression compared with CD56brightNK
cells. Thus, miR-155 expression does not inversely correlate with
the constitutive expression of its target SHIP1 in CD56dimand
CD56brightNK cells, suggesting that SHIP1 is probably regulated
via miR-155–dependent and –independent mechanisms in these
NK subsets as has been previously observed for SHIP1 in other
hematopoietic tissues42and as noted in this section. Further,
CD56brightand CD56dimNK cells represent stage 4 and stage 5 of
human NK cell development, respectively.43Whether miR-155
may also target the 3? UTR of genes important for the transition
between stage 4 and stage 5 of NK cell development remains to be
We showed that SHIP1 is down-modulated in primary human
NK cells after costimulation with IL-12 and IL-18,13and under
these conditions we observed an inverse correlation between
miR-155 and SHIP1 expression. We prove that miR-155 is down
modulating SHIP1 by recapitulating this effect in NK-92 cells
ectopically expressing an anti–miR-155, and by amplifying the
effect by overexpressing miR-155. These experiments, together
with our previous finding that SHIP1 regulates IFN-? in both
human cells and in mouse NK cells in vitro and in vivo,13identify
the inositol phosphatase SHIP1 as one of the intermediaries
through which miR-155 exerts its biologic effect on NK cell IFN-?
NK cells mediate ADCC largely through CD16, and this
receptor has been the focus of intense research throughout the
years. It has been shown by Parihar et al8that CD16 activation in
the presence of IL-12 induces IFN-? expression in a synergistic
fashion in human NK cells. Collaboratively, we also found that
SHIP1 was a negative regulator of IFN-? in both mouse and human
NK cells under similar conditions.16Our findings in this study are
in agreement with this, wherein CD16 activation induces miR-155,
which is synergistically enhanced by the addition of IL-12. These
results suggest that IL-12 primes NK cells for the induction of
miR-155 not only via the aforementioned up-regulation of
IL-18R? but also through means that affect other signaling
pathways, such as the colocalization of IL-12R and CD16 to cell
lipid rafts and synergistic activation of extracellular signal-
regulated kinase (ERK).44In fact, activation of BIC and miR-155
expression by B-cell receptor signaling occurs through the ERK
SHIP1 is down-modulated by CD16 activation in NK cells,
implicating its involvement in this regulation of IFN-?. In contrast
to the combination of IL-12 and IL-18, we did not observe
synergistic down-modulation of SHIP1 protein with IL-12 and
CD16 activation, having seen a near-maximal down modulation
with CD16 activation alone. Taken with the previous finding that
IL-12 synergized with CD16 activation for IFN-? production,8and
with our current finding that IL-12 significantly enhanced CD16-
mediated miR-155 expression, it is probable that (1) activation of
CD16 regulates SHIP1 via both miR-155–dependent and –independent
mechanisms, and (2) miR-155 enhances IFN-? production by targeting
more than just SHIP1. The latter notion is consistent with the fact that
miR-155 has several targets23and that the regulation of IFN-? gene
In human primary NK cells and NK-92 cells ectopically
overexpressing miR-155, we did not observe any effect on
spontaneous cytotoxicity or ADCC (data not shown). There is still
the possibility that long-term culture in IL-2 during infection of
primary NK cells and NK-92 cells may compensate for an effect of
miR-155 on NK cell cytotoxicity which may have otherwise been
The data from the current study suggest a potential model for
the induction of miR-155 and the role of miR-155 in regulating
IFN-? production in NK cells after NK cell activation. The
induction of miR-155 expression depends on signaling events
induced by triggering the IL-18R or CD16, but not directly via the
IL-12 receptor. The combination of IL-12 with either IL-18 or
CD16 activation induces miR-155 in a synergistic fashion. The
synergistic induction of miR-155 expression after IL-12 and
IL-18 costimulation of NK cells depends on the induction of
IL-18R? by IL-12 stimulation whereas the synergistic induction of
miR-155 by IL-12 and CD16 stimulation possibly depends on
increasing signaling downstream of CD16. In turn miR-155
overexpression targets the 5? inositol phosphatase SHIP1 with
consequent up-regulation of the phosphatidylinositol-3 kinase
pathway and subsequent enhanced IFN-? production (Figure 8).
In summary, we show that miR-155 plays a central role in NK
cell IFN-? production, whether through monokine signaling or
CD16 activation. This is accomplished at least in part by directly
regulating the expression of SHIP1, although our data suggest that
miR-155 probably targets other components in parallel. By gaining a
IFN-?, we may be able to identify new targets that can control
chronic inflammation and/or modulate NK cell antitumor activity.
The authors thank Susan McClory and Ed Briercheck for helpful
Figure 8. Schematic representation modeling the induction of miR-155 after NK
cell activation and its role in regulating IFN-? production. miR-155 expression
was induced after activation of resting NK cells via IL18 alone, via CD16 activation
alone, but not via IL-12 alone; however, the combination of IL-12 with either IL-18 or
CD16 activation induced miR-155 in a synergistic fashion. This synergism was
dependent on the induction of the IL-18R by IL-12, whereas the synergistic induction
of miR-155 noted with CD16 and IL-12 possibly results from enhanced intracellular
signaling downstream of CD16 (dashed arrow). miR-155 targets the 5? inositol
phosphatase SHIP1, thereby down-modulating SHIP1 expression, which in turn
promotes the prolonged activation of the PI3K pathway and subsequent enhanced
production of IFN-?.
3484 TROTTAet alBLOOD, 12APRIL 2012?VOLUME 119, NUMBER 15
For personal use only.on November 5, 2015. by guest
This work was supported in part by the National Cancer
Institute grants CA16058, CA95426, and CA68458 (all M.A.C.).
Contribution: R.T. designed the study, performed research work,
analyzed data, and wrote the paper; L.C., D.C., and S.J.
performed experimental work and analyzed data; C.M. per-
formed cell sorting experiments; L.Y. analyzed data; S.C.,
J.P.B., S.T., and C.M.C. provided reagents and discussed data;
and M.A.C. contributed to the design of the study and to the
writing/editing of the paper.
Conflict-of-interest disclosure: The authors declare no compet-
ing financial interests.
Correspondence: Rossana Trotta or Michael A. Caligiuri, The
Ohio State University Comprehensive Cancer Center, 884 and
886 OSU Biomedical Research Tower, 460 West 12th Ave,
Columbus, OH 43210; e-mail: firstname.lastname@example.org or
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miR-155 IN NK CELLS3485BLOOD, 12APRIL 2012?VOLUME 119, NUMBER 15
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online February 29, 2012
2012 119: 3478-3485
Lianbo Yu, Jonathan P. Butchar, Susheela Tridandapani, Carlo M. Croce and Michael A. Caligiuri
Rossana Trotta, Li Chen, David Ciarlariello, Srirama Josyula, Charlene Mao, Stefan Costinean,
production in natural killer cells
miR-155 regulates IFN-
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