Group Title: Pediatric Rheumatology 2008, 6 (Suppl 1): P159
Title: The Pompe Registry: tracking Pompe disease symptoms in a broad patient population
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Title: The Pompe Registry: tracking Pompe disease symptoms in a broad patient population
Series Title: Pediatric Rheumatology 2008, 6 (Suppl 1): P159
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Prasad S
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Available online http://arthritis-research.com/content/10/4/R101


Research article

Upregulated miR-146a expression in peripheral blood

mononuclear cells from rheumatoid arthritis patients
Kaleb M Pauley1, Minoru Satoh2,3, Annie L Chan2, Michael R Bubb2, Westley H Reeves2,3 and
Edward KL Chan1


1Department of Oral Biology, University of Florida, SW Archer Road, Gainesville, Florida 32610, USA
2Division of Rheumatology and Clinical Immunology, Department of Medicine, University of Florida, SW Archer Road, Gainesville, Florida 32610, USA
3Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, Gainesville, SW Archer Road, Florida 32610, USA

Corresponding author: Edward KL Chan, echan@ufl.edu

Received: 29 May 2008 Revisions requested: 26 Jun 2008 Revisions received: 8 Aug 2008 Accepted: 29 Aug 2008 Published: 29 Aug 2008

Arthritis Research & Therapy 2008, 10:R101 (doi:10.1186/ar2493)
This article is online at: http://arthritis-research.com/content/1 0/4/R101
C 2008 Pauley et al.; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Abstract


Introduction MicroRNAs are small noncoding RNA molecules
that negatively regulate gene expression via degradation or
translational repression of their targeted mRNAs. It is known that
aberrant microRNA expression can play important roles in
cancer, but the role of microRNAs in autoimmune diseases is
only beginning to emerge. In this study, the expression of
selected microRNAs is examined in rheumatoid arthritis.

Methods Total RNA was isolated from peripheral blood
mononuclear cells obtained from patients with rheumatoid
arthritis, and healthy and disease control individuals, and the
expression of miR-146a, miR-155, miR-132, miR-16, and
microRNA let-7a was analyzed using quantitative real-time PCR.

Results Rheumatoid arthritis peripheral blood mononuclear
cells exhibited between 1.8-fold and 2.6-fold increases in miR-
146a, miR-155, miR-132, and miR-16 expression, whereas let-
7a expression was not significantly different compared with
healthy control individuals. In addition, two targets of miR-146a,
namely tumor necrosis factor receptor-associated factor 6
(TRAF6) and IL-1 receptor-associated kinase 1 (IRAK-1), were
similarly expressed between rheumatoid arthritis patients and
control individuals, despite increased expression of miR-146a in


Introduction
Rheumatoid arthritis (RA) is a systemic autoimmune disorder
that is characterized by chronic inflammation of synovial tissue,
which results in irreversible joint damage [1]. Inflammatory


patients with rheumatoid arthritis. Repression of TRAF6 and/or
IRAK-1 in THP-1 cells resulted in up to an 86%/ reduction in
tumor necrosis factor-a production, implicating that normal miR-
146a function is critical for the regulation of tumor necrosis
factor-a production.


Conclusions Recent studies have shown that synovial tissue
and synovial fibroblasts from patients with rheumatoid arthritis
exhibit increased expression of certain microRNAs. Our data
thus demonstrate that microRNA expression in rheumatoid
arthritis peripheral blood mononuclear cells mimics that of
synovial tissue/fibroblasts. The increased microRNA expression
in rheumatoid arthritis patients is potentially useful as a marker
for disease diagnosis, progression, or treatment efficacy, but
this will require confirmation using a large and well defined
cohort. Our data also suggest a possible mechanism
contributing to rheumatoid arthritis pathogenesis, whereby miR-
146a expression is increased but unable to properly function,
leading to prolonged tumor necrosis factor-a production in
patients with rheumatoid arthritis.


cytokines, including tumor necrosis factor (TNF)-a and IL-1 p,
play an important role in RA pathogenesis, and inhibition of
these cytokines can ameliorate disease in some patients [2,3].


Page 1 of 10
(page number not for citation purposes)


CRP: C-reactive protein; ESR: erythrocyte sedimentation rate; GWP: GW or P bodies; IL: interleukin; IFN: interferon; IIF: indirect immunofluores-
cence; IRAK: IL-1 receptor-associated kinase; LPS: lipopolysaccharide; MCP: monocyte chemoattractant protein; M-CSF: macrophage colony-stim-
ulating factor; miRNA: microRNA; PBMC: peripheral blood mononuclear cell; qRT-PCR: quantitative real-time RT-PCR; RA: rheumatoid arthritis;
RISC: RNA-induced silencing complex; RT-PCR: reverse transcription polymerase chain reaction; siRNA: small interfering RNA; TNF: tumor necrosis
factor; TRAF: tumor necrosis factor receptor-associated factor.







Arthritis Research & Therapy Vol 10 No 4 Pauley et al.



MicroRNAs (miRNAs) are small noncoding RNA molecules
that negatively regulate gene expression at the post-transcrip-
tional level [4,5]. It is predicted that as much as one-third of all
mRNAs are targeted for miRNA-mediated regulation [6], and
the importance of miRNA regulation is becoming increasingly
clear as new roles in critical cellular processes such as apop-
tosis, differentiation, and the cell cycle are discovered.

The biogenesis and maturation of miRNAs are dependent on
two RNase III enzymes, namely Drosha and Dicer. First, miR-
NAs are transcribed by RNA polymerase II into a long primary
miRNA (pri-miRNA) transcript [7,8]. The pri-miRNA is then
cleaved by Drosha and its partner protein DGCR8 into an
approximately 70-nucleotide precursor miRNA (pre-miRNA)
molecule [9-13]. The pre-miRNA is then exported into the
cytoplasm via Exportin 5, where it is cleaved into an approxi-
mately 21-nucleotide miRNA duplex, similar in structure to
small interfering RNA (siRNA) [14,15]. One strand of the
miRNA duplex is then loaded into the RNA-induced silencing
complex (RISC), where it binds the 3'-untranslated region of
its target mRNA, causing the degradation or translational
repression of that mRNA [15].

The key components of RISC are the argonaute proteins 1-4
(Agol-4). Ago2 is known to be the catalytic enzyme of RNA
interference and is critical for both miRNA and siRNA function
[16,17]. In addition to Ago2, many other proteins are critical
for miRNA function, including GW182 and Rck/p54. These
proteins, as well as miRNA and siRNA, localize to cytoplasmic
foci known as GW or P bodies (here referred to as GWB). Our
recent studies have established GWB to be useful biomarkers
for siRNA and miRNA activity in cells [18,19]. Our latest study
(Pauley KM and coworkers, unpublished data) demonstrated
that the number and size of GWB significantly increases con-
currently with increased miRNA expression in lipopolysaccha-
ride (LPS)-treated THP-1 cells, implying that GWB can be
monitored as biomarkers for miRNA activity.

TNF-a stimulation has been shown to induce the expression of
certain miRNAs, including miR-146a and miR-155, in mono-
cytes and macrophages [20,21]. Based on these data and the
fact that TNF-a plays an important role in RA pathogenesis, as
supported by the development of successful anti-TNF-a ther-
apies, we set out to compare miRNA expression between RA
patients and healthy control individuals.

In this study, we obtained peripheral blood mononuclear cells
(PBMCs) from RA patients and control individuals and exam-
ined the expression of miR-146a, miR-155, miR-132, miR-16,
and miRNA let-7a. Most of these miRNAs were chosen for
examination based on previous reports linking them to immune
stimulation by LPS or TNF-a; miR-1 6 was selected for its abil-
ity to target the 3'-untranslated region of TNF-a [20-22].
miRNA let-7a was chosen as a control. This study is significant
because it demonstrates that miRNA expression in RA


PBMCs may mimic conditions in synovial tissue and thus ena-
ble us to bypass the need for synovial tissue samples, allowing
the analysis of larger patient populations.

Materials and methods
Patients and control individuals
Sixteen patients (including two samples from a single patient)
who fulfilled the American College of Rheumatology classifica-
tion criteria for RA were included in the study. Their demo-
graphic, clinical, and laboratory characteristics are
summarized in Table 1. Four disease control individuals,
including one with systemic lupus erythematosus, two with
Sjdgren's syndrome, and one with systemic sclerosis, were
included. Nine healthy donors with no history of autoimmune
disease were included as control individuals. This study was
approved by the University of Florida Institutional Review
Board, and written permission was obtained from all who par-
ticipated in the study.

PBMC collection and quantitative real-time RT-PCR
Blood samples were collected in EDTA-treated tubes and
PBMCs were isolated by standard Ficoll density-gradient cen-
trifugation. PBMCs were washed once in sterile phosphate-
buffered saline (PBS) before culture or RNA isolation. Total
RNA was isolated from freshly obtained PBMCs using the mir-
Vana miRNA Isolation kit (Ambion, Austin, TX, USA), in
accordance with the manufacturer's protocol. RNA concentra-
tions were determined and 10 ng of each RNA sample were
used for quantitative real-time RT-PCR (qRT-PCR). miRNA
qRT-PCR was performed using the TaqMan MicroRNA
Reverse Transcription Kit, TaqMan Universal PCR Master Mix,
and TaqMan MicroRNA Assay primers for human miR-146a,
miR-155, miR-132, miR-16, and miRNA let-7a (Applied Bio-
systems, Foster City, CA, USA). mRNA qRT-PCR was per-
formed using the TaqMan High-Capacity cDNA Reverse
Transcription Kit, TaqMan Fast PCR Master Mix, and TaqMan
mRNA assay primers (Applied Biosystems). All reactions were
analyzed using StepOne Real-Time PCR System (Applied
Biosystems). The levels of miRNA were normalized to U44
controls, whereas mRNA levels were normalized to 18S RNA.
The cycle threshold (Ct) values, corresponding to the PCR
cycle number at which fluorescence emission reaches a
threshold above baseline emission, were determined and the
relative miRNA or mRNA expression was calculated using the
2-Act method [23].

Cell culture and cytokine treatment
THP-1 human monocytes obtained from American Type Cul-
ture Collection (Manassas, VA, USA) were cultured in RPMI
1640 medium with 2 mmol/l L-glutamine, 4.5 g/l glucose, 10
mmol/l HEPES, 1.0 mmol/l sodium pyruvate, 0.05 mmol/l 2-
mercaptoethanol, and 10% fetal bovine serum. THP-1 cells
were seeded at 5 x 105 cells per well in a six-well plate and
treated with 10 ng/ml TNF-a, IFN-y, IL-12p70, IL-4, IL-10 (BD
Biosciences, San Jose, CA, USA), IFN-a, IFN-P (PBL Inter-


Page 2 of 10
(page number not for citation purposes)








Available online http://arthritis-research.com/content/10/4/R101


Table 1

Demographic, clinical, and laboratory information of patients


Subject

RA Patients

RA-1 aa
RA-lb
RA-2

RA-3


RA-6

RA-7

RA-8
RA-9


RA-10
RA-11
RA-12

RA-13
RA-14
RA-15
RA-16

Disease control individuals
SLE1


SSc1


Sex Age (years) Medications


Male 65
Male 65
Female 33

Female 43
Female 45
Female 55
Female 73

Female 51
Male 61
Male 46

Female 32
Female 50
Male 67

Female 55
Female 33
Female 55
Female 56


Female 36
Female 30

Male 21
Female 57


CRP (mg/1) ESR (mm/hour)


None


4.14b 9


Etanercept, naproxen

Etanercept
None


None

Hydroxychloroquine, MTX, prednisone

Prednisone, MTX, hydroxychloroquine, sulfasalazine
Methylprednisolone, MTX, etanercept
Leflunomide
Etanercept, MTX

MTX, celecoxib, prednisone


Hydroxychloroquine, ibuprofen
None

Prednisone, minocycline


No data No data


4.8 26
2.1 30

No data 15
No data 1


No data No data
0.6 11

No data 17
2.8 7
168.2 88
No data No data


Hydroxychloroquine, prednisone, mycophenolate mofetil

Hydroxychloroquine, mycophenolate mofetil

Hydroxychloroquine
Cyclosporine, hydroxychloroquine, prednisone,
mycophenolate mofetil


aSample collected from RA-1 before and after MTX treatment. bNormal value less than 0.8. The normal CRP is <4.9 mg/I, and the normal ESR is
<20 mm/hour for females and <10 mm/hour for males. CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; MTX, methotrexate; RA,
rheumatoid arthritis; SLE, systemic lupus erythematosus; SjS, Sjogren's syndrome; SSc, scleroderma.


feron Source, New Brunswick, NJ, USA), or macrophage col-
ony-stimulating factor (M-CSF; US Biological, Swampscott,
MA, USA). Cells were also treated with 25 ng/ml monocyte
chemoattractant protein (MCP)-1 (Sigma) in serum-free
media. After the designated treatment time had elapsed, cells
were harvested and washed once in PBS before analysis.

Indirect immunofluorescence
THP-1 cells were cytospun onto glass slides at 1,000 rpm for
5 minutes. PBMCs were cultured on glass slides at 370C for
1 hour. Cells were fixed in 3% paraformaldehyde for 10 min-
utes and permeabilized in 0.5% Triton X-100 for 5 minutes.
GWB were detected in THP-1 cells with a human prototype
anti-GWB serum [24] used at 1:6,000 dilution, and in PBMCs
with rabbit anti-Rck/p54 antibodies used at 1:500 dilution.
TNF receptor-associated factor (TRAF)6 and IL-1 receptor-
associated kinase (IRAK)-1 were detected using rabbit anti-


TRAF6 (1:50; Santa Cruz Biotechnology, Santa Cruz, CA,
USA) and rabbit anti-IRAK-1 (1:50; Santa Cruz Biotechnol-
ogy). Secondary antibodies used were Alexa Fluor 488 goat
anti-human IgG or goat anti-rabbit IgG (1:400) from Molecular
Probes (Carlsbad, CA, USA). Slides were mounted using
Vectashield Mounting Medium with 4',6-diamidino-2-phenylin-
dole (DAPI; VECTOR Laboratories, Burlingame, CA, USA).
Fluorescence images were taken with Zeiss Axiovert 200 M
microscope and a Zeiss AxioCam MRm camera using the 20x
or 40 x 0.75 NA objectives. Color images were assessed
using Adobe Photoshop version 7 (Adobe Systems Inc., San
Jose, CA, USA). GWB were counted using Cell-Profiler image
analysis software [25].

siRNA transfection
siRNAs targeting TRAF6 and IRAK-1 were transfected into
THP-1 cells using Lipofectamine 2000 (Invitrogen, Carlsbad,


Page 3 of 10
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Arthritis Research & Therapy Vol 10 No 4 Pauley et al.



CA, USA), in accordance with the manufacturer's instructions.
To monitor the transfection efficiency, Cy3-labeled siRNA tar-
geting lamin A/C was transfected into cells in parallel in all
transfections, and at least 80% transfection efficiency was
achieved. The siRNAs used in this study were all purchased
from Applied Biosystems. The sense and antisense strand
sequences were as follows: IRAK-1: 5'-GGUUUCGUCAC-
CCAAACAUtt-3' and 5'-AUGUUUGGGUGACGAAACCtg-
3'; TRAF6: 5'-GGUUGUUUGCACAAGAUGGtt-3' and 5'-
CCAUCUUGUGCAAACAACCtt-3'.

Multiplex analysis of cytokines
THP-1 cells were transfected as described above and then
treated with 1 tg/ml LPS (Salmonella enterica serotype min-
nesota; Sigma, St. Louis, MO, USA) for 24 hours in culture
medium. The culture supernatant was then harvested and fro-
zen at -800C for storage before multiplex analysis. The human
cytokine/chemokine LINCOplex premixed kit (LINCO
Research, St. Charles, MO, USA) was used in accordance
with the manufacturer's protocol in order to detect human
MCP-1 and TNF-a quantitatively.

Results and discussion
Specific cytokines/chemokines induce GWB in THP-1
cells and human PBMCs
Since our previous work demonstrated that GWB can be used
as biomarkers for miRNA activity (Pauley KM, unpublished
data), we began to examine a variety of cytokines and chemok-
ines for their ability to stimulate miRNA activity in human mono-
cytic THP-1 cells. THP-1 cells were treated with 10 ng/ml
TNF-a, IFN-a, IFN-P, IFN-y, IL-12p70, M-CSF, IL-4, IL-10, or
25 ng/ml MCP-1 for 4 hours. Indirect immunofluorescence
(IIF) was performed using a human anti-GWB serum to detect
GWB in the cells. As shown in Figure 1, the proinflammatory
cytokines/chemokines TNF-a, IFN-a, IFN-P, IFN-y, and MCP-1
resulted in a significant increase in the number of GWB per
cell compared with untreated cells cultured in parallel (P <
0.0001, as determined using one-way analysis of variance).
However, IL-12p70, M-CSF, IL-4, and IL-10 had no significant
effect on the number of GWB. TNF-a elicited the strongest
response in THP-1 cells, with fourfold increase in the average
number of GWB per cell (Figure la,c). These experiments
were repeated at least three times, with reproducible results
each time.

Next, we decided to examine the effect of TNF-a stimulation
on human PBMC GWB. GWB staining using human PBMCs
from a healthy donor, after 4 hours stimulation with TNF-a (1
ng/ml), is shown. Similar to THP-1 cells, the number of GWB
per cell increased 3.5-fold after TNF-a stimulation of PBMCs
(Figure 1b,c; P < 0.0001, as determined by Mann Whitney
test). These data indicated that THP-1 cells may be suitable
substitutes for human PBMCs in some of the subsequent
experiments.


Figure 1


35
S30
25
20
i 15
0 10
5


THP-1


THP-1


(b) PBMC


PBMC


untreated









TNF-a


TNF-a treatment results in increased number of GWB in THP-1 and
human PBMCs. (a) THP-1 cells were treated with 10 ng/ml TNF-u,
IFN-u, IFN-p, IFN-y, IL-1 2p70, M-CSF, IL-4, IL-10, or 25 ng/ml MCP-1
for 4 hours. IIF was performed using a human anti-GWB serum to
detect GWB, and the number of GWB were counted using CellProfiler
image analysis software. Average number of GWB per cell and SEM is
shown. *P < 0.0001, as determined by one-way analysis of variance.
(b) Human PBMCs were obtained from a healthy donor and isolated
using Ficoll density-gradient centrifugation. The cells were then cul-
tured for 4 hours in the presence of 1 ng/ml TNF-c. GWB were
detected by IIF using rabbit anti-Rck/p54 antibodies. Average number
of GWB and SEM is shown. *P< 0.0001, as determined by Mann-
Whitney test. (c) IIF image of THP-1 and PBMCs treated with 10 ng/ml
or 1 ng/ml TNF-a for 4 hours, respectively. GWB are shown in green,
and nuclei are counterstained with 4',6-diamidino-2-phenylindole
(DAPI; blue). Bar= 10 p.m. GWP, GW or P bodies; IL, interleukin; IFN,
interferon; IIF, indirect immunofluorescence; MCP, macrophage chem-
oattractant protein; M-CSF, macrophage colony-stimulating factor;
PBMC, peripheral blood mononuclear cell; SEM, standard error of the
mean; TNF, tumor necrosis factor.


RA patient PBMCs exhibit increased expression of miR-
146a, miR-155, miR-132, and miR-16
In Figure 1 we showed that TNF-a is a potent inducer of GWB
and therefore miRNA activity. Our preliminary studies and
work from other investigators have confirmed that TNF-a stim-
ulation induces the expression of certain miRNAs, including


Page 4 of 10
(page number not for citation purposes)


_T_


R~"~a;9~tss?,a~""; 4;












miR-146a and miR-155 [20,21]. Based on these data and the
important role played by TNF-a in RA pathogenesis and ther-
apies, we began to investigate the expression levels of miRNA
in RA patients as compared with those in healthy and disease
control individuals. PBMCs were obtained from patients (n =
17 RA patients and n = 4 disease control individuals) and
healthy donors (n = 9) and isolated by Ficoll density-gradient
centrifugation. Initially, RA PBMCs were monitored by IIF for
GWB; however, we did not observe an increased number of
GWB in RA compared with healthy control individuals (not
shown). This discrepancy could be due to limited sensitivity in
the quantitation of GWB.

As shown in Figure 2a, the average relative expression levels
of miR-146a, miR-155, miR-132, and miR-16 were 2.6-, 1.8-,
2.0-, and 1.9-fold, respectively, higher for RA patients than for
healthy control individuals (P < 0.01 for miR-146a and P <
0.05 for miR-155, miR-132, and miR-16, as determined by
one-way analysis of variance). The expression of miRNA let-7a
was not significantly different between RA patients and
healthy control individuals (Figure 2a). Disease control miRNA
expression resembled that in healthy control individuals.

To examine the relationship between RA disease activity and
miRNA expression levels, patients were classified into inac-
tive/remission and active patients, based on C-reactive protein
(CRP) and erythrocyte sedimentation rate (ESR) values. Three
patients with normal CRP and ESR (Table 1) were classified
as inactive, whereas eight patients with elevated CRP and/or
ESR (Table 1) were classified as active (Figure 2b). Those
patients with incomplete or no available data were omitted
(Table 1). miRNA expression levels were compared between
the groups. Interestingly, high miR-146a and miR-16 expres-
sion levels appeared to correlate with active disease, whereas
low expression level correlated with inactive disease (Figure
2b; P < 0.05, as determined by t-test). These data indicates
that miR-146a and miR-16 expression levels may be a useful
marker of RA disease activity. Further studies involving a larger
patient cohort are needed to determine fully whether monitor-
ing miRNA expression as a marker for disease activity can
improve upon CRP or ESR measurements.

Figure 2c shows the miRNA expression levels in two samples
from a single RA patient collected over a 2-month interval, dur-
ing which time this patient's CRP and ESR values increased
despite methotrexate treatment. The miRNA levels of this
patient were largely unchanged over the 2-month interval,
remaining elevated compared with those in healthy control
individuals. This indicates that the elevated miRNA expression
in this patient may reflect the patient's lack of improvement, as
indicated by the increased CRP and ESR values. In this
patient, miR-146a, miR-155, and miR-132 expression levels
were stable over this time period, whereas the expression lev-
els of miR-16 and miRNA let-7a increased by approximately
3.5-fold and 2.4-fold, respectively. A larger patient population


Available online http://arthritis-research.com/content/10/4/R101



must be examined in order to determine whether miRNA
expression levels may be indicative of treatment efficacy.

To further analyze the increased miRNA expression exhibited
by these RA patients, we compared miR-146a, miR-155, and
miR-132 expression levels with patient clinical and demo-
graphic data (Table 1) and found no significant trends or cor-
relations between high expression levels and age, race, or
medications. Patients receiving no medications at the time of
miRNA analysis exhibited the same trend toward elevated
miRNA expression, indicating that treatment with medications
is not responsible for the increased miRNA expression in RA
patients.

Recently, two reports [26,27] showed increased miR-146 and
miR-155 expression levels in RA synovial tissue and fibrob-
lasts. Stanczyk and coworkers [27] reported a fourfold
increase in miR-146a expression and a twofold increase in
miR-155 expression in RA synovial fibroblasts compared with
osteoarthritis synovial fibroblasts. They also demonstrated that
miR-155 expression can repress the induction of matrix metal-
loproteinases 3 and 1, indicating that miR-155 may be
involved in modulating the destructive properties of RA syno-
vial fibroblasts. However, in that report, miR-155 expression
from RA PBMCs was not significantly different from that in
control PBMCs. This discrepancy could be due to differences
in experimental techniques or patient populations. Nakasa and
colleagues [26] also reported an approximately fourfold
increase in miR-146a expression in RA synovial tissue. Our
data demonstrate that RA patient PBMCs exhibit elevated
miRNA expression in a similar manner to RA synovial tissue,
with a 2.6-fold increase in miR-146a expression and a 1.8-fold
increase in miR-155 expression. Because of the invasiveness
involved in collecting samples, monitoring miRNA expression
in RA synovial tissue is, in most cases, limited to extremely
severe disease in patients undergoing joint surgery or replace-
ment. Because blood collection is not invasive, this allows for
easy sample collection over time, which is a distinct advantage
when monitoring disease activity and treatment efficacy.

Monocyte/macrophage population of RA PBMCs
exhibits increased miRNA expression
Because PBMCs are composed of a mixed cell population,
the two main components of which are monocytes/macro-
phages and lymphocytes, we wished to determine which cell
population in RA patients exhibits increased miRNA expres-
sion. PBMCs were isolated from RA patients (n = 2) and incu-
bated in tissue culture dishes at 370C for 1 hour. The
monocyte/macrophage population adhered to the dish,
whereas lymphocytes remained in suspension. The adherent
cells were washed five times with sterile PBS, and the nonad-
herent cells were collected and washed with sterile PBS. The
purity of the adherent population was approximately 80%, as
determined by microscopy. RNA was isolated from the cells,
miRNA expression was analyzed by qRT-PCR, and the data


Page 5 of 10
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Arthritis Research & Therapy Vol 10 No 4 Pauley et al.


Figure 2


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RA patients exhibit aberrant expression of miR-146a, miR-155, miR-132 and miR-16 versus healthy controls. (a) RNA was isolated from healthy con-
trol individuals (n = 9), disease control individuals (n = 4), and RA patient (n = 17). PBMCs and relative expression levels of miR-146a, miR-155,
miR-132, miR-16, and miRNA let-7a were analyzed by qRT-PCR using U44 RNA as an internal control. Average is indicated by bars. *P < 0.05, **P
< 0.01, as determined by one-way analysis of variance. For RA patients, closed circles indicate patients undergoing anti-TNF-c therapy at time of
sample collection, squares indicate MTX treatment, and open circles indicate other or no treatment. (b) Disease activity was determined for patients
using CRP and ESR values and correlated with miRNA expression. Normal CRP and ESR values were classified as inactive disease (n = 3; patients
9, 12, and 14 in Table 1), and higher than normal CRP or ESR values were classified as active disease (n = 8; patients 1a, 1b, 2, 4, 5, 6, 10, and 15
in Table 1). Those patients with no or incomplete data for CRP/ESR values were omitted. *P < 0.05, as determined by t-test. (c) PBMGs were col-
lected from patient RA-1 before (November 2007) and after (January 2008) MTX treatment and miRNA expression was examined using qRT-PCR.
miRNA expression is largely consistent over time, with the exception of increased miR-16 expression. CRP, C-reactive protein; ESR, erythrocyte
sedimentation rate; miRNA, microRNA; MTX, methotrexate; PBMC, peripheral blood mononuclear cell; qRT-PCR, quantitative real-time RT-PCR;
RA, rheumatoid arthritis; RT-PCR, reverse transcription polymerase chain reaction; TNF, tumor necrosis factor.


Page 6 of 10
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o other/none
aMTX
* TNFa


f-f












were normalized within the total group of patient and control
samples.

The expression levels of miR-146a, miR-155, miR-132, and
miR-1 6 were 2.8-, 1.6-, 4.2-, and 3.4-fold higher, respectively,
in monocytes than in lymphocytes (Figure 3a). Let-7a expres-
sion was similar between monocytes and lymphocytes (not
shown). Figure 3b shows the average expression (miR-146a,
miR-155, miR-132, and miR-1 6 combined) for the monocyte
and lymphocyte populations of two RA patients (P < 0.02, as
determined by Mann-Whitney test). These findings suggest
that monocytes/macrophages contribute to the increased
miRNA expression observed in RA patients more than lym-
phocytes, but further studies must be performed to confirm
this observation.

TRAF6 and IRAK-1 expression is similar between RA
patients and control individuals
Because most of the RA patients exhibited increased expres-
sion of miR-146a compared with healthy and disease control
individuals, we decided to examine the expression of two con-
firmed targets of miR-146a, namely TRAF6 and IRAK-1 [21].
TRAF6 and IRAK-1 mRNA expression levels were analyzed by
qRT-PCR (Figure 4a,b). RA patients exhibited increased miR-
146a production compared with control individuals, and we
therefore expected to observe decreased TRAF6 and/or
IRAK-1 expression in RA patients as compared with control
individuals. However, TRAF6 and IRAK-1 mRNA expression
levels were very similar between RA patients and control indi-

Figure 3


(b)

I-monocytes
Mlymphocytes


Monocyte/macrophage fraction of PBMCs exhibit increased miRNA
expression compared with lymphocyte fraction. PBMCs were collected
from RA patients and separated into monocyte/macrophage and lym-
phocyte populations by allowing the monocytes/macrophages to
adhere to a tissue culture dish. (a) miRNA expression was examined
using qRT-PCR. SEM is shown (n = 2 patients). (b) Average expres-
sion levels of miR-146a, miR-155, miR-132, and miR-16 are shown for
monocyte and lymphocyte populations for two RA patients. *P < 0.02,
as determined by Mann-Whitney test. SEM is shown. PBMC, peripheral
blood mononuclear cell; qRT-PCR, quantitative real-time RT-PCR; RA,
rheumatoid arthritis; RT-PCR, reverse transcription polymerase chain
reaction; SEM, standard error of the mean.


Available online http://arthritis-research.com/content/10/4/R101



viduals, and overall the mRNA levels of TRAF6 and IRAK-1 did
not exhibit the same degree of variability between patients that
we observed with miRNA expression. This may indicate that
TRAF6 and IRAK-1 transcripts are under other levels of con-
trol. To confirm this discrepancy, we analyzed TRAF6 and
IRAK-1 protein levels by IIF in one healthy control individual

Figure 4


TRAF6 (b) IRAK-1


00 CV0
?IP~
:No :S''


c-=healthy ctl
RA


TRAF6 and IRAK-1 expression levels are similar between RA patients,
healthy controls, and disease controls. RNA was isolated from PBMCs
from healthy control individuals (n = 9), disease control individuals (n =
4) and RA patients (n = 14), and mRNA expression levels of (a) TRAF6
and (b) IRAK-1 were analyzed using qRT-PCR. (c) PBMCs isolated
from a healthy control individual and RA patient were incubated on
glass slides for 1 hour at 37C. The adhered cells were fixed and per-
meabilized in 3% paraformaldehyde and 0.5% Triton X-1 00, respec-
tively. Protein levels of TRAF6 and IRAK-1 were analyzed by
immunofluorescence using rabbit anti-TRAF6 and anti-IRAK-1 antibod-
ies, and relative fluorescence was determined using Image J analysis
software. SEM is shown; n > 20 cells. IRAK, IL-1 receptor-associated
kinase; PBMC, peripheral blood mononuclear cell; qRT-PCR, quantita-
tive real-time RT-PCR; RA, rheumatoid arthritis; RT-PCR, reverse tran-
scription polymerase chain reaction; SEM, standard error of the mean;
TRAF, tumor necrosis factor receptor-associated factor.


Page 7 of 10
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Vd







Arthritis Research & Therapy Vol 10 No 4 Pauley et al.



and one RA patient whose miR-146a level was increased (Fig-
ure 2a). PBMCs were processed for IIF as previously
described and were stained for TRAF6 and IRAK-1. Image J
software was used to quantify the relative level of fluorescence
for at least 20 cells. As shown in Figure 4c, there was no sig-
nificant difference in TRAF6 or IRAK-1 protein levels between
the RA patient and healthy control individual, which is consist-
ent with the mRNA analysis.

It is interesting to speculate that this lack of regulation of
TRAF6/IRAK-1 by miR-146a could play a role in RA pathogen-
esis, especially because it has been reported that inhibition of
IRAK-1 using antisense oligonucleotides results in decreased
LPS-induced cytokine production [28], and our preliminary
data have shown that transfection of miR-146a into THP-1
monocytes results in knockdown of TRAF6 and IRAK-1
expression and inflammatory cytokine production (Pauley KM
and coworkers, unpublished data). To investigate this possibil-
ity further, we transfected siRNA targeting TRAF6 and/or
IRAK-1 into THP-1 cells. The knockdown efficiency was deter-
mined by analyzing TRAF6 and IRAK-1 mRNA levels by qRT-
PCR, and at least 80% and 60% knockdown was achieved for
TRAF6 and IRAK-1, respectively (Figure 5a). Two days after
transfection, knockdown and control cells were treated with 1
tg/ml LPS for 24 hours. Culture supernatants were collected
and cytokines/chemokines were quantitatively detected using
a human cytokine multiplex assay. TNF-a production was dras-
tically reduced in the TRAF6 and/or IRAK-1 deficient cells
compared with mock transfected cells (Figure 5b), whereas
MCP-1 production was not affected by TRAF6 or IRAK-1
knockdown (Figure 5c).

These data demonstrate that TRAF6 and IRAK-1 are required
for the production of TNF-a in THP-1 cells. Taken together, it
is reasonable to hypothesize that the absence of TRAF6/IRAK-
1 regulation by miR-146a in RA patients could contribute to
the prolonged production of TNF-a that many of these patients
exhibit. Furthermore, it would be interesting to investigate the
expression patterns of miR-146a, TRAF6, and IRAK-1 in RA
patients who are responsive to anti-TNF-a therapy versus
those who are not responsive. Clearly, further studies are
needed to elucidate the role played by miR-146a regulation in
RA pathogenesis and the mechanism by which TRAF6/IRAK-
1 escape miR-146a regulation.

Conclusion
In summary, this study demonstrates that PBMCs from RA
patients exhibit statistically significant increased expression
levels of miR-146a, miR-155, miR-132, and miR-16 compared
with healthy and disease control individuals. Furthermore, we
demonstrated that high levels of miR-146a and miR-16
expression correlate with active disease, whereas low expres-
sion levels correlate with inactive disease. Although miR-146a
expression is increased in RA patients, levels of the two estab-
lished miR-146a targets TRAF6 and IRAK-1 in RA patients are


Figure 5

(a)

S1.5. -=TRAF6 expression
*2 IRAK-1 expression
S1.0-


0.5-

in
1 I I a
"


TNF-a


12-
10-
8-
6-
6
4-
2-


MCP-1
- r_1


Knockdown of TRAF6 and/or IRAK-1 results in decreased TNF-a pro-
duction in THP-1 cells. THP-1 cells were transfected with siRNA tar-
geting TRAF6 and/or IRAK-1. (a) 48 hours after transfection, mRNA
levels of TRAF6 and IRAK-1 were analyzed by qRT-PCR and normal-
ized to mock transfected cells. SEM shown (n = 2). After knockdown of
TRAF6 and/or IRAK-1 was confirmed by qRT-PCR, cells were treated
with 1 gpg/ml LPS for 24 hours and culture supernatants were col-
lected. Multiplex assay was used to quantitatively detect (b) TNF-c and
(c) MCP-1. IRAK, IL-1 receptor-associated kinase; LPS, lipopolysac-
charide; MCP, macrophage chemoattractant protein; PBMC, peripheral
blood mononuclear cell; qRT-PCR, quantitative real-time RT-PCR; RT-
PCR, reverse transcription polymerase chain reaction; TNF, tumor
necrosis factor; TRAF, tumor necrosis factor receptor-associated
factor.

similar to those in control individuals. We also show that
TRAF6 and IRAK-1 regulation is important for TNF-a produc-
tion in THP-1 cells.

Normally, stimuli such as LPS or TNF-a will induce expression
of miR-146a, miR-155, and miR-132 in a nuclear factor-KB
dependent manner [21]. In the case of miR-146a, this will lead
to negative regulation of TRAF6 and IRAK-1, which in turn will
decrease the production of proinflammatory cytokines/chem-


Page 8 of 10
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okines, including TNF-a. Thus, the function of miR-146a, at
least in part, is to control the extent of the stimulation, such that
the production of some of these proinflammatory cytokines/
chemokines will not continue for an extended period of time.
However, it is interesting to speculate that defective negative
regulation of TRAF6 or IRAK-1 by the increased miR-146a in
RA patients is the cause of prolonged TNF-a production.

Although it is very exciting that two independent studies have
shown increased miRNA expression in RA synovial tissue,
analyzing miRNA expression in patient PBMCs presents a dis-
tinct advantage over analyzing synovial tissue samples. Collec-
tion of PBMCs is noninvasive, and samples can be collected
from patients ranging in disease severity from early onset to
more severe, whereas synovial tissue collection is biased
toward patients with severe degenerative disease. With fur-
ther validations and studies conducted in larger patient popu-
lations, monitoring of selected miRNAs could prove to be a
valuable addition to RA diagnostics, or monitoring disease
progression or treatment efficacy. The underlying mechanisms
resulting in increased miRNA expression and inability of miR-
146a to regulate its targets need to be elucidated, and these
mechanisms may be potential targets for the development of
new RA therapies.


Competing interests
The authors declare that they have no competing interests.

Authors' contributions
KMP designed and conducted all experiments and drafted the
manuscript. MS assisted with statistical evaluations, provided
clinical insights, and edited the manuscript. ALC collected
patient samples. MRB and WHR recruited study subjects, and
provided clinical insights and advice. EKLC conceived of the
study, assisted in designing the study, and edited the manu-
script. All authors read and approved the final manuscript.

Acknowledgements
This work was supported in part by NIH Grants A147859, AR051 766
and M01 R00082. KMP is supported by NIDCR oral biology training
grant T32 DE007200.

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