Content uploaded by Frieda A Koopman
Author content
All content in this area was uploaded by Frieda A Koopman on Jul 11, 2016
Content may be subject to copyright.
Vagus nerve stimulation inhibits cytokine production
and attenuates disease severity in rheumatoid arthritis
Frieda A. Koopman
a
, Sangeeta S. Chavan
b
, Sanda Miljko
c
, Simeon Grazio
d
, Sekib Sokolovic
e
, P. Richard Schuurman
f
,
Ashesh D. Mehta
g
, Yaakov A. Levine
h
, Michael Faltys
h
, Ralph Zitnik
h
, Kevin J. Tracey
b
, and Paul P. Tak
a,1,2,3,4
a
Amsterdam Rheumatology and Immunology Center, Department of Clinical Immunology and Rheumatology, Academic Medical Center, University of
Amsterdam, 1105 AZ Amsterdam, The Netherlands;
b
Laboratory of Biomedical Science, Feinstein Institute for Medical Research, Manhasset, NY 11030;
c
University Clinical Hospital, Mostar 88000, Bosnia and Herzegovina;
d
Clinical Hospital Center Sestre Milosrdnice, Zagreb 10000, Croatia;
e
Sarajevo
University Clinical Center, Sarajevo 71000, Bosnia and Herzegovina;
f
Department of Neurosurgery, Academic Medical Center, University of Amsterdam,
1105 AZ Amsterdam, The Netherlands;
g
Department of Neurosurgery, Hofstra Northwell School of Medicine, Manhasset, NY 11030; and
h
SetPoint Medical
Corporation, Valencia, CA91355
Edited by Ruslan Medzhitov, Yale University School of Medicine, New Haven, CT, and approved June 1, 2016 (received for review April 18, 2016)
Rheumatoid arthritis (RA) is a heterogeneous, prevalent, chronic
autoimmune disease characterized by painful swollen joints and
significant disabilities. Symptomatic relief can be achieved in up to
50% of patients using biological agents that inhibit tumor necrosis
factor (TNF) or other mechanisms of action, but there are no univer-
sally effective therapies. Recent advances in basic and preclinical
science reveal that reflex neural circuits inhibit the production of
cytokines and inflammation in animal models. One well-characterized
cytokine-inhibiting mechanism, termed the “inflammatory reflex,”is
dependent upon vagus nerve signals that inhibit cytokine produc-
tion and attenuate experimental arthritis severity in mice and rats. It
previously was unknown whether directly stimulating the inflam-
matory reflex in humans inhibits TNF production. Here we show that
an implantable vagus nerve-stimulating device in epilepsy patients
inhibits peripheral blood production of TNF, IL-1β,andIL-6.Vagus
nerve stimulation (up to four times daily) in RA patients significantly
inhibited TNF production for up to 84 d. Moreover, RA disease se-
verity, as measured by standardized clinical composite scores, im-
proved significantly. Together, these results establish that vagus
nerve stimulation targeting the inflammatory reflex modulates
TNF production and reduces inflammation in humans. These findings
suggest that it is possible to use mechanism-based neuromodulating
devices in the experimental therapy of RA an d possibly othe r a u-
toimmune and autoinflammatory diseases.
vagus nerve
|
rheumatoid arthritis
|
inflammatory reflex
|
tumor necrosis factor
|
cytokines
Rheumatoid arthritis (RA) is a chronic inflammatory disease
characterized by synovial inflammation in the musculoskeletal
joints resulting in cartilage degradation and bone destruction with
consequent disability (1). The prevalence exceeds 1.3 million adult
cases in the United States, with attributable medical costs estimated
between $19–39 billion (2, 3). Standard therapies include gluco-
corticoids, methotrexate, monoclonal antibodies, and other phar-
macological agents targeting inflammatory mechanisms (4). Despite
these treatment options, many RA patients fail to respond, instead
persisting with poor health, shortened life span, and significant
impairments in quality of life affecting work, leisure, and social
functions (5, 6). Thus, there remains a significant need for al-
ternative therapeutic approaches.
Recent advances at the intersection of immunology and neuro-
science reveal reflex neural circuit mechanisms regulating innate
and adaptive immunity (7, 8). One well-characterized reflex circuit,
termed the “inflammatory reflex,”is defined by signals that travel in
the vagus nerve to inhibit monocyte and macrophage production of
tumor necrosis factor (TNF) and other cytokines (7). Electrical
stimulation of the vagus nerve in animals (e.g., mouse, rat, and dog)
stimulates choline acetyltransferase-positive T cells to secrete ace-
tylcholine in spleen and other tissues (9). Acetylcholine is the cog-
nate ligand for α-7 nicotinic acetylcholine receptors (α7nAChR)
expressed on cytokine-producing monocytes, macrophages, and
stromal cells (7, 10, 11). Ligand binding inhibits the nuclear trans-
location of NF-κB and inhibits inflammasome activation in mac-
rophages activated by exposure to lipopolysaccharide (LPS), other
Toll-like receptor (TLR) ligands, and other proinflammatory
stimulating factors (12, 13).
Inflammatory reflex signaling, which is enhanced by electrically
stimulating the vagus nerve, significantly reduces cytokine pro-
duction and attenuates disease severity in experimental models of
endotoxemia, sepsis, colitis, and other preclinical animal models of
inflammatory syndromes (7, 8, 14–16). In experimental collagen-
induced arthritis, vagotomy or selective disruption of α7nAChR
worsened disease severity, and administration of nicotine or other
selective α7nAChR agonists, ameliorated disease severity (17, 18).
Vagus nerve stimulation delivered once daily for 60 s with an
Significance
Rheumatoid arthritis (RA) is a chronic, prevalent, and disabling au-
toimmune disease that occurs when inflammation damages joints.
Recent advances in neuroscience and immunology have mapped
neural circuits that regulate the onset and resolution of in-
flammation. In one circuit, termed “the inflammatory reflex,”action
potentials transmitted in the vagus nerve inhibit the production of
tumor necrosis factor (TNF), an inflammatory molecule that is a
major therapeutic target in RA. Although studied in animal models
of arthritis and other inflammatory diseases, whether electrical
stimulation of the vagus nerve can inhibit TNF production in hu-
mans has remained unknown. The positive mechanistic results
reported here extend the preclinical data to the clinic and reveal
that vagus nerve stimulation inhibits TNF and attenuates disease
severity in RA patients.
Author contributions: F.A.K., S.S.C., S.M., S.G., S.S., P.R.S., A.D.M., Y.A.L., M.F., R.Z., K.J.T.,
and P.P.T. designed research; F.A.K., S.S.C., S.M., S.G., S.S., P.R.S., A.D.M., Y.A.L., R.Z., K.J.T.,
and P.P.T. per formed research; F .A.K., S.S .C., S.M., S.G., S .S., P.R.S., A .D.M., Y.A.L ., M.F., R.Z.,
K.J.T., and P.P.T. analyzed data; and F.A.K., S.S.C., S.M., S.G., S.S., P.R.S., A.D.M., Y.A.L., M.F.,
R.Z., K.J.T., and P.P.T. wrote the paper.
Conflict of interest statement: M.F., Y.A.L., and R.Z. are employees of and equity holders
in SetPoint Medical Corporation. K.J.T. is an equity holder in and has received consulting
fees from SetPoint Medical Corporation. S.M., S.G., S.S., P.R.S., and P.P.T. received research
grants from SetPoint Medical Corporation to support the c linical study reported here.
P.P.T. has rec eived consulting f ees from SetPoint Med ical Corporatio n and is currently
an employee of GlaxoSmithKline, which holds an equity interest in SetPoint
Medical Corporation.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
1
Present address: Department of Medicine, University of Cambridge, Cambridge CB2 1TN,
United Kingdom.
2
Present address: Department of Rheumatology, Ghent University, 9000 Ghent, Belgium.
3
Present address: Research & Development, GlaxoSmithKline, Stevenage SG1 2NY,
United Kingdom.
4
To whom correspondence should be addressed. Email: P.P.Tak@amc.uva.nl.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1605635113/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1605635113 PNAS Early Edition
|
1of6
IMMUNOLOGY AND
INFLAMMATION
implanted device attenuated joint swelling, inhibited cytokine pro-
duction, and conferred significant protection against synovitis and
periarticular bone erosions (19, 20). Accordingly, we reasoned that
it might be possible to modulate cytokine levels and inflammation
using an active implantable medical device in humans (20).
Vagus nerve-stimulating devices have been used for decades in
patients with refractory epilepsy and have been used more recently
in patients with depression. These devices have been implanted in
more than 100,000 patients, are relatively well tolerated, and have
not been associated with immunosuppression or long-term com-
plications (21, 22). We implanted a cohort of epilepsy patients with
a vagus nerve-stimulating device and observed that transient de-
livery of electrical current during general anesthesia significantly
inhibited TNF production in peripheral blood monocytes. A sub-
sequent study of 17 RA patients in an 84-d open-label trial also
revealed significantly decreased TNF production and significantly
improved clinical signs and symptoms of disease.
Results
To determine whether vagus nerve stimulation inhibits TNF pro-
duction in humans, we studied seven epilepsy patients [five male,
two female; mean age 35 y (range 25–43 y)] who were implanted
with a vagus nerve-stimulating device using a coiled cuff electrode
(Cyberonics) on the left cervical vagus nerve. These patients had no
history of inflammatory or autoimmune disorders. Peripheral blood
was collected before, during, and after vagus nerve stimulator
implantation surgery. Endotoxin was added to the whole blood to
stimulate the production of TNF by monocytes for 4 h (13, 23). The
application of current-controlled electrical pulses (single 30-s stim-
ulation at 1.0-mA output current, 20-Hz pulse frequency, 500-μs
pulse duration) significantly inhibited whole-blood TNF production
compared with baseline levels before electrical stimulation (Fig.
1A). The inhibition of TNF release following vagus nerve stimula-
tion during general anesthesia cannot be attributed to a placebo
effect, because the subjects were unconscious and were not aware of
the nerve stimulation. Whole-blood production of interleukin (IL)-6
and IL-1βwas also inhibited significantly by vagus nerve stimulation
(Fig. 1 Band C). To our knowledge, this is the first report that the
delivery of electric current applied directly on the cervical vagus
nerve to stimulate the inflammatory reflex inhibits the endotoxin-
induced release of TNF, IL-1β, and IL-6 in humans.
We next studied the effects of vagus nerve stimulation in patients
with RA. At enrollment the 18 study patients had active dis-
ease, with at least four tender and four swollen joints (of a 28-joint
count), despite methotrexate therapy for at least 3 mo on a stable
dose. One patient from cohort I, who fulfilled the American College
of Rheumatology (ACR)/European League Against Rheumatism
(EULAR) classification for RA, was later diagnosed with Whipple
disease and was excluded from the efficacy analysis. This patient is
included in the baseline patient characteristics (Table 1) and ad-
verse-event data (Table S1). The RA patients with active disease
were studied in two cohorts. Cohort I (n=7) included patients with
active disease despite therapy with methotrexate. They had never
received a biological TNF antagonist or had previously failed
treatment with TNF antagonists because of drug toxicity. Cohort II
(n=10) included patients who had failed conventional therapy with
methotrexate and also had failed treatment with at least two bi-
ological agents differing in mechanisms of action (e.g., anti-TNF,
anti–IL-6 receptor, anti-CD20 antibodies, and/or T-cell costim-
ulation inhibitor). There were no deaths, serious adverse events,
withdrawals from the study because of adverse events, or infections
in either cohort. In agreement with known risks of the procedure,
nine patients experienced mild or moderate adverse events associ-
ated with implanting the vagus nerve stimulator on the left cervical
vagus nerve (Table S1).
The study design schematic is shown in Fig. 2A. The vagus nerve
was stimulated during surgery (day −14) to measure electrode im-
pedance and to verify device function. During the 14-d post-
operative recovery period (day −14 to day 0), the device was turned
off, and no current was delivered to the vagus nerve. On the first
treatment day (study day 0), patients received a single 60-s stimu-
lation with electric current pulses of 250-μs duration at 10 Hz and
an output current between 0.25–2.0 mA, as tolerated. No further
stimulation was delivered for 7 d. On study day 7, the output current
was adjusted to the highest amperage tolerated, up to 2.0 mA; this
level of current was subsequently delivered once daily for 60 s in
250-μs pulse widths at 10 Hz. Current escalation up to the highest
tolerated amperage (up to 2.0 mA) was repeated weekly until day
28. At that visit the frequency of daily stimulation events was in-
creased to four times daily in patients who had not achieved a
moderate or good clinical response according EULAR criteria (24).
On day 28, the output current delivered was comparable in both
cohorts: cohort I output current was 1.29 ±0.37 mA (mean ±SD);
cohort II output current was 1.60 ±0.36 mA. In cohort I, two of
seven patients received electric current pulses four times daily.
In cohort II, 6/10 patients received electric current pulses four
times daily.
We observed that TNF production in cultured peripheral blood
obtained from the combined RA study cohort on day 42 was sig-
nificantly reduced from baseline day −21 (TNF =2,900 ±566 pg/mL
on day −21 vs. 1,776 ±342 pg/mL on day 42, P<0.05) (Fig. 2B). On
day 42 the vagus nerve stimulator was turned off. After a 14-d hiatus,
it was restarted on day 56, and patients were followed through day
84. After the vagus nerve stimulator was turned off, TNF production
Pre-Anesthesia
Post-Anesthesia
Pre-VNS
4Hr Post-VNS
0
400
800
1200
TNF (pg/mL)
*
*
IL-6 (pg/mL)
Pre-Anesthesia
Post-VNS
0
200
400
600
800
**
IL-1 (pg/mL)
Pre-Anesthesia
Post-VNS
0
50
100
150
200
250
*
A
BC
Fig. 1. Inflammatory reflex activation reduces whole-blood LPS-induced TNF
production in epilepsy patients. Electrical stimulation of the vagus nerve in hu-
mans inhibits whole-blood LPS-induced TNF release. Blood was obtained from
epilepsy patients (n=7) undergoing implantation of a vagus nerve-stimulation
device at different time points: before anesthesia induction and before vagus
nerve stimulation; after anesthesia induction and before vagus nerve stimulation
(pre-VNS); and 4 h after vagus nerve stimulation (post-VNS). Whole blood was
incubatedwithLPSandTNF(A), IL-6 (B), and IL-1β(C) levels in plasma were de-
termined after 4 h in culture. The significance of the differences between mean
values at each time point was tested by unpaired ANOVA (*P<0.05, **P<0.01).
Data are shown as mean ±SEM.
2of6
|
www.pnas.org/cgi/doi/10.1073/pnas.1605635113 Koopman et al.
increased significantly by day 56; when the stimulator was turned on
again, TNF production again decreased significantly by day 84
(1,776 ±342 pg/mL on day 42 vs. 2,617 ±342 pg/mL on day 56
and 1,975 ±407 pg/mL on day 84, P<0.01 for both). This finding
indicates that active electrical stimulation of the vagus nerve in-
hibits TNF production in patients with RA.
RA signs and symptoms are measured using a standard disease
activity composite score [the 28-joint C-reactive protein (CRP)-
based disease activity score, DAS28-CRP] derived from counting
swollen joints and tender joints, a patient-defined visual analog
score of disease activity, and serum CRP levels (25). We ob-
served that DAS28-CRP values at day 42 were significantly im-
proved (i.e., lower) from baseline day −21 in the combined
cohorts (DAS28-CRP =6.05 ±0.18 on day −21 vs. 4.16 ±0.39
on day 42, P<0.001), when the device was delivering current
(Fig. 2C). Within days after receiving electrical stimulation of the
vagus nerve, the DAS28-CRP improved significantly in some
patients (Fig. S1). When the device was turned off (at day 42), the
DAS28-CRP increased significantly within 14 d (4.16 ±0.39 on day
42 vs. 4.96 ±0.31 on day 56, P=0.001). Restarting the device (day
56) significantly reduced the DAS28-CRP (Fig. 2C). Linear re-
gression analysis comparing the mean change in the DAS28-CRP
and the percentage change in TNF release from baseline day −21
to day 42 revealed a highly significant correlation (r=0.384, P<
0.0001) (Fig. 2D). The temporal pattern of TNF production in
the combined cohort correlated with the DAS28-CRP (Fig. 2E).
We assessed the fraction of patients who improved from baseline
to achieve ACR 20%, 50%, and 70% clinical responses and also the
number of patients who improved from baseline sufficiently to meet
the definition for EULAR response and remission. The ACR re-
sponse is defined as the percentage improvement in disease activity
between two time points (ACR20 is ≥20%, ACR 50 is ≥50%, and
ACR70 is ≥70% improvement). The EULAR response depends on
the change in the DAS28-CRP and the absolute level achieved after
treatment (24). As shown in Fig. S2, the ACR and EULAR re-
sponse criteria were fulfilled in a large subset of patients in both
cohorts. At the primary endpoint (day 42) the percentages of pa-
tients fulfilling the ACR response criteria for 20%, 50%, and 70%
improvement were 71.4%, 57.1%, and 28.6%, respectively, for co-
hort I and were 70.0%, 30.0%, and 0.0%, respectively, for cohort II.
The percentages of patients achieving DAS28 remission (DAS28-
CRP <2.6) on day 42 in cohorts I and II were 28.6% and 0.0%,
respectively. Improvement was observed in all constituent compo-
nents of the composite end points (tender joint count, swollen joint
count, patient’s assessment of pain, patient’s global assessment,
physician’s global assessment, and CRP) (Table S2). Together,
these data indicate that vagus nerve stimulation inhibits TNF
and significantly attenuates RA disease severity.
We measured a panel of serum cytokines to assess further the
mechanisms of this experimental therapeutic intervention. Most,
including serum TNF, IL-10, IL-12p70, IL-13, IL-1α,IL-1β,IL-2,
IL-4, IL-5, and TNF-β, were below 1 pg/mL (unreliable limits of
detection). Serum IL-6 levels in subjects who improved by EULAR
criteria were significantly decreased compared with subjects who
failedtoimprove:IL-6levelswere15.4±2.4 pg/mL in nonre-
sponders (n=5) vs. 5.0 ±1.4 pg/mL in responders (n=12) (P=
0.001) (Fig. 3A). Decreased IL-6 levels in the patients who
responded to therapy correlated with improvement in disease se-
verity between day −21 and day 42 (r=0.707, P=0.002) (Fig. 3B).
The IL-6 responses are specific, because IL-8 and IL-17 levels did
not change significantly [IL-8: 25.6 ±9.1 pg/mL in nonresponders
(n=5) vs. 13.7 ±1.7 pg/mL in responders (n=12), P=0.29 (Fig.
3C); IL-17: 2.8 ±1.1 pg/mL in nonresponders (n=5) vs. 1.8 ±
0.2 pg/mL in responders (n=12), P=0.18 (Fig. 3E)] and did
not correlate to clinical response (Fig. 3 Dand F).
Discussion
To our knowledge, this study is the first to assess whether stimulating
the inflammatory reflex by directly implanting an electronic device
modulates TNF and other cytokines in humans. Historically the de-
velopment of electrically active implantable medical devices has been
primarily empiric, based upon observing effects of devices that deliver
Table 1. RA patient baseline demographics, medication history, and disease severity
Demographics Cohort I Cohort II Combined
Total, n81018
Enrollment by country
Bosnia 3 0 3
Croatia 2 0 2
The Netherlands 3 10 13
Mean age in years (range) 55 (36–69) 48 (36–56) 51 (36–69)
Sex, % female 50 100 78
Ethnicity, % Caucasian 88 100 94
Mean no. of years since RA diagnosis (SD) 9.9 (5.7) 11.8 (6.3) 11.0 (5.9)
No. rheumatoid factor-positive patients (%) 7 (88) 5 (50) 12 (67)
No. anti-citrullinated peptide Ab
+
patients (%) 6 (75) 6 (60) 12 (67)
No. patients receiving prior nonbiologic disease-modifying antirheumatic drugs (%)
0 drugs 1 (13) 1 (10) 2 (11)
1 drug 2 (25) 3 (30) 5 (28)
2 drugs 2 (25) 2 (20) 4 (22)
3 or more drugs 3 (37) 4 (40) 7 (39)
No. patients receiving prior biologic disease-modifying antirheumatic drugs (%)
0 drugs 3 (38) 0 3 (17)
1 drug 4 (50) 0 4 (22)
2 drugs 1 (12) 0 1 (6)
3 drugs 0 3 (30) 3 (17)
4 drugs 0 4 (40) 4 (22)
5 drugs 0 2 (20) 2 (11)
6 drugs 0 1 (10) 1 (6)
DAS28-CRP (SD) 6.05 (0.87) 5.94 (0.72) 5.99 (0.77)
High-sensitivity CRP, mg/L (SD) 17.5 (10.0) 17.5 (18.5) 17.5 (14.9)
Koopman et al. PNAS Early Edition
|
3of6
IMMUNOLOGY AND
INFLAMMATION
electrical current to depolarize neuronal or cardiac tissue. Absent
appropriate biomarkers or mechanistic understanding, it has been
difficult or impossible to develop or optimize the device parameters
for current delivery, physiological effect in the targeted organ system,
and clinical efficacy. Direct and accessible surrogate molecular
markers of disease mechanism targeted by active implantable
medical devices are uncommon. The discovery of the inflammatory
reflex affords a unique opportunity for developing a neuromodulating
device to regulate immune cell function by targeting a neural pathway
that regulates cytokine production, a surrogate marker of molecular
mechanism (26).
RA patients in cohort I are in early stages of disease not
responding to therapy with methotrexate. These patients are fre-
quently candidates for subsequent therapy with a biological agent
that inhibits TNF. Cohort II patients are in later stages of disease,
having failed multiple biological disease-modifying antirheumatic
drugs. After electrical stimulation of the vagus nerve the DAS28-
CRP improved significantly in both cohorts, and withdrawal of
treatment significantly worsened the severity of disease. Reactivating
the device on day 56 restored significant clinical improvement. The
clinical responses were accompanied by significant reductions in
TNF release during periods of disease remission and significant in-
creases in TNF release during disease exacerbation. A large body of
preclinical evidence has delineated the molecular and physiological
mechanisms of the inflammatory reflex modulating TNF, IL-6,
HMGB1, and other cytokines (7–9, 11–20). The molecular mecha-
nisms of cytokine inhibition implicate acetylcholine derived from
T
ChAt
cells, the subset of choline acetyltransferase-positive T cells that
we identified in the inflammatory reflex (9). In future clinical trials it
should be interesting to study whether T
ChAt
cells participate in
mediating anti-inflammatory reflex mechanisms.
Vagus nerve stimulation has been used to treat medically re-
fractory epilepsy in more than 100,000 patients, and it is generally
well tolerated (21, 22). The adverse events reported here were mild
to moderate in severity and were comparable in type and frequency
to those seen in prior studies of vagus nerve stimulation therapy in
epilepsy patients. These adverse events included transient hoarse-
ness, postoperative hoarseness from neuropraxis, and transient
intraoperative bradycardia during surgery. None of the patients
developed infection. Larger clinical trials can be designed to
-21 0 42 56 84
0
1000
2000
3000
4000
Study Visit Day
TNF (pg/mL)
*
*
^
+
#
-3.0
-2.5
-2.0
-1.5
-1.0
-0.5
0.0
-21-14 0 7 14 28 42 56 8421
Study Visit Day
Mean Change in DAS28-CRP
Cohort I (N = 7)
Cohort II (N = 10)
Combined (N = 17)
*
+
#
^
*
+
//
*
Treatment Hiatus
Primary Endpoint
Treatment Hiatus
Implantation and
Diagnostic Stimulus
^
0 100 200 300 400
-6
-4
-2
0
2
TNF Release (% Change from Day -21)
Change in DAS28-CRP from Day -21
r=0.3855, **** p < 0.0001
-3
-2
-1
0
0
1000
2000
3000
Study Visit Day
Mean Changein DAS28-CRPfromDay -21
TNF(pg/mL;TC)
DAS
TNF
Primary Endpoint
Implantation and
Diagnostic Stimulus
Treatment Hiatus Treatment Hiatus
-21-14 0 7 14 21 28 42 56 84
A
BC
DE
Baseline Clinical
Assessments
Daily
Stimulation
Escalating
Intensity
QID
Stimulation
if Clinical
Response
Inadequate
Primary Endpoint
D-21 D-14 D0 D7 D14 D21 D28 D42 D56 D84
Intrao perative stim ulation
during diagnostic check
Single in -clinic
stimulatio n
Discontinu e
stimulation
Restart stimulation at
same level as D42
Fig. 2. The effects of inflammatory reflex activation on whole-blood LPS-induced TNF production and disease activity in RA patients. (A) Schematic of the RA study
design. D −21 to D84 indicate study visit days. The stimulation schedule and timing of assessments are shown. (B) Mean LPS-induced TNF production in the combined
RA cohort (n=17) at study days −21, 0, 42, 56, and 84; visit means are designated by bars, and error bars indicate SEM. Differences in means were tested for
significance by paired ttest: *P<0.05 vs. d −21;
+
P<0.01 vs. d 0;
^
P<0.01 vs. d 42;
#
P<0.01 vs. d 56. (C) The mean change in DAS2 8-CRP from baseline by study visit
day for cohort I (patients failing methotrexate treatment), cohort II (patients failing treatment by multiple biologic agents), and combined cohorts. The significance
of the mean change by paired ttest between visits is shown: *P<0.05 vs. d −21;
//
P<0.01 vs. d −21;
+
P<0.001 vs. d −21;
#
P<0.001 vs. d 42; ^P<0.05 vs. d 42). (D)
Linear regression analysis comparing the changes in the DAS28-CRP and the percent change in TNF release from study day −21 measured at each individual visit for
each patient in the combined cohort. Changes in the DAS28-CRP and TNF release are significantly correlated by Pearson’stest(r=0.384, P<0.0001). (E) Mean
change in the DAS28-CRP and mean LPS-induced TNF release over time by study visit day. Changes in the DAS28-CRP and TNF release follow a similar temporal
pattern in response to initial simulation, stimulation withdrawal, and stimulation reinitiation.
4of6
|
www.pnas.org/cgi/doi/10.1073/pnas.1605635113 Koopman et al.
determine the risk/benefit ratio for implantable electronic devices
compared with the toxicity and side effects of pharmacological and
targeted therapies for RA.
The electrical stimulation parameters used in this study were
previously established to stimulate the inflammatory reflex in pre-
clinical studies and differ significantly from the stimulation proto-
cols used in epilepsy (19, 27). Here, electrical current (up to
2.0 mA) was delivered to the cervical vagus nerve for 60 s one to
four times daily; the maximum time of electrical current flow for any
patient in this study was 4 min daily. This stimulation protocol
differs significantly from the protocols for treating epilepsy, in which
current (up to 2.25 mA) is delivered at 60-s intervals, followed by an
OFF interval of 5–180 min, repeated continuously. Thus, epilepsy
patients may receive electrical current delivery for up to 240 min
daily. Preclinical studies have established that stimulation of the
inflammatory reflex for as little as 60 s confers significant inhibition
of cytokine production for up to 24 h. The present study was not
designed or powered to evaluate the relationship between specific
electrical current dose–response and clinical outcomes or the
longer-term durability of therapeutic benefit, and the effects of
under- or overstimulation of the inflammatory reflex are also an
important area for future study.
The primary objective of this study was to determine whether
activating the inflammatory reflex with an implanted electronic
device inhibits cytokine production in humans. It is reasonable to
consider whether placebo mechanisms contribute to these find-
ings, because some patients are aware when the device is de-
livering current. There are several arguments against a placebo
effect explaining the observed inhibition of TNF and IL-6 and
the significant clinical improvements. First, we observed that
intraoperative vagus nerve stimulation significantly inhibited
TNF release in epilepsy patients who were unconscious during
the implantation. These patients could not be aware of the
stimulation, indicating that the suppression of cytokine release
immediately following vagus nerve stimulation cannot be at-
tributed to a placebo effect. Second, we also observed that the
suppression of TNF release during vagus nerve stimulation in
RA patients occurred only when the device was functioning. It
has been established previously that biomarkers are not modifiable
by placebo effects in RA studies of this duration (28, 29). Third, we
observed reduced TNF and IL-6 production and positive clinical
responses in the subset of therapy-resistant patients who had failed
both methotrexate therapy and treatment with multiple biologic
agents with differing mechanisms of action. It has been established
in prior studies that placebo response rates in drug-resistant cohorts
are extremely low (ACR20 responses 5–11%). The findings here of
significantly higher ACR20 responses (between 70% and 71.4%)
argue strongly against a placebo effect being the mechanism.
Fourth, a recent study reported clinical improvement using vagus
nerve-stimulation therapy to treat another disease mediated by
TNF, Crohn’s disease (30). Although the investigators in that study
did not measure the activity of the inflammatory reflex or cytokine
production, they did examine endoscopic biopsies and observed that
vagus nerve stimulation significantly inhibited inflammation in the
colonic tissues, an objective histological tissue response that cannot
be attributed to placebo effects. Finally, our recent prospective
observational studies indicate that impaired constitutive vagus nerve
activity precedes the development of clinically manifest RA (31).
Therefore, when considered together with extensive preclinical data
that identify molecular and neurophysiological mechanisms, the
inhibition of TNF during electrical stimulation and the signifi-
cant clinical responses shown give evidence that the clinical mech-
anism is mediated by the inflammatory reflex.
This first-in-class study supports a conceptual framework for
further studies of electronic medical devices in diseases currently
treated with drugs, an approach termed “bioelectronic medicine”
(32). Larger clinical trials in RA can be designed and powered to
assess clinical efficacy, but our findings encourage pursuing this
strategy in RA and in other cytokine-mediated autoimmune and
auto-inflammatory disorders.
Materials and Methods
Study of Vagus Nerve Stimulation in Epilepsy Patients. The study of vagus nerve
stimulation in epilepsy patients was performed at the Hofstra Northwell School of
Medicine and was approved by the Clinical Research Center (CRC) and the In-
stitutional Review Board. All patients provided informed consent before partici-
pation. The study population consisted of seven epilepsy patients being implanted
with a Cyberonics Vagus Nerve Stimulation System (Cyberonics) according to the
manufacturer’s instructions as part of their standard care for the treatment of
refractory epilepsy (Fig. S3). During the intraoperative diagnostic procedure, the
pulse generator produces a 30-s stimulation at a 1.0-mA output current with pulse
frequency of 20 Hz and pulse width of 500 μs. Blood samples were taken before
anesthesia induction, after anesthesia induction but before intraoperative vagus
nerve stimulation, and 4 h after intraoperative vagus nerve stimulation.
The LPS-induced cytokine release assay was performed as previously described
(33). Cytokine levels were analyzed using the MSD multiplex cytokine assay
(Meso Scale Discovery) per the manufacturer’s instructions. TNF, IL-6, and IL-1β
Nonresponder Responder
0
5
10
15
20
25
IL-6 (pg/mL;D42)
**
-5-4-3-2-101
0
5
10
15
20
25
DAS28 (D-21 to D42)
IL-6 (pg/mL;D42)
Nonresponder Responder
0
20
40
60
IL-8 (pg/mL;D42)
-5-4-3-2-101
0
20
40
60
DAS (D-21 to D42)
IL-8 (pg/mL;D42)
Nonresponder Responder
0
2
4
6
8
IL-17 (pg/mL;D42)
-5-4-3-2-101
0
2
4
6
8
DAS (D-21 to D42)
IL-17(pg/mL;D42)
AB
CD
EF
Fig. 3. Modulation of serum cytokines. Serum from each patient in the com-
bined cohort was analyzed for multiple analytes at day 42. (A,C,andE) Indi-
vidual patient values for EULAR nonresponders and responders are shown for IL-6
(A), IL-8 (C), and IL-17 (E) levels. The significance of differences between
mean values at each time point was tested by unpaired ttest (**P<0.01).
Horizontal bars indicate mean ±SEM. (B,D, and F) Linear regression analysis
comparing analyte level at day 42 to the change in the DAS28-CRP from
study day −21 to day 42. The change in the DAS28-CRP is significantly cor-
related to IL-6 release (r=0.707, P=0.002) (B) but not to IL-8 release (r=
0.261, P=0.31) (D) or IL-17 release (r=0.384, P=0.07) (F).
Koopman et al. PNAS Early Edition
|
5of6
IMMUNOLOGY AND
INFLAMMATION
release across time points was analyzed using the Prism analytical software
package (GraphPad).
Study of Vagus Nerve Stimulation in RA Patients. The study of vagus nerve
stimulation in RA patients was performed at one center in The Netherlands (the
Academic Medical Center of the University of Amsterdam), at two centers in
Bosnia and Herzegovina (the University Clinical Hospital in Mostar and Sarajevo
University Clinical Center in Sarajevo), and at in one center in Croatia (Clinical
Hospital Center Sestre Milosrdnice, Zagreb) and was approved by the respective
national and institutional Ethics Committees. All patients provided informed
consent before participation. The investigational study device was a Cyberonics
Vagus Nerve Stimulation System, implanted as described above. The systems were
treated as investigational study devices because of their off-label use in patients
with RA. The study recruited two separate patient cohorts. Cohort I consisted of
RA patients who had failed to respond to methotrexate and who were either TNF-
antagonist naive or had previously failed treatment with a TNF antagonist be-
cause of safety reasons rather than lack of efficacy. Cohort II included patients
who had not responded adequately to at least two biologic agents with at least
two different mechanisms of action. Major inclusion and exclusion criteria are
given in SI Materials and Methods. The use of prednisone at a stable daily dose
of less than 10 mg and other nonbiological disease-modifying antirheumatic
drugs at stable doses was allowed.
The design schematic of this single-arm study is shown in Fig. 2A.Atthe
conclusion of the study, patients were offered the options of having the device
surgically removed or left in place and inactivated or continuing treatment in a
long-term extension study. All recruited subjects opted to continue in the ex-
tension study, which will be reported separately.
The primary study end point was mean change in the DAS28-CRP between visits
on baseline day −21 and day 42 (25). Mean changes in the DAS28-CRP between
day −21 and day 42 or day 84, and between day 42 and day 56 also were assessed
for significance at P<0.05 using a Student’spairedttest in the SAS 9.2 statistical
analysis package (SAS). Because this was an exploratory study, no formal statistical
power calculations were performed, and no adjustments for multiple comparisons
were made. Adverse events were collected from the day of implantation through
the day 84 visit, coded using the Medical Dictionary for Regulatory Activities
(MedDRA), and presented by MedDRA term as subject incidence rates.
Whole-Blood Cytokine Release Assay in the RA Study. The TruCulture system
(Myriad RBM), an assay system suitable for use at clinical sites and scalable to larger
studies, was used. Venous blood was drawn into tubes containing endotoxin at
100 ng/mL and was incubated at 37 °C for 24 h. Supernatant TNF was measured by
ELISA (R&D Systems). Comparisons of changes in TNF release between baseline and
subsequent visits [with three statistical outlier exclusions; robust regression and
outlier removal (ROUT) method with the maximum false discovery rate at 1%] by
paired ttest and linear regression analysis of relationships between changes
in the DAS28-CRP and TNF release were performed using the Prism analytical
software package.
Serum Cytokines in the RA Study. Serum cytokine levels from day 42 were an-
alyzed using the MSD multiplex cytokine assay as above. Analysis of IL-6, IL-8, and
IL-17 release on day 42 and linear regression analysis of relationships (Pearson’s
test) between the change in the DAS28-CRP and serum cytokine release at
day 42 were performed using the Prism analytical software package.
ACKNOWLEDGMENTS. We thank the patients for their participation,
Dr. Martin Lesser for his biostatistical input, and April Caravaca and Anna
Drake for technical assistance.
1. Smolen JS, Aletaha D, McInnes IB (May 3, 2016) Rheumatoid arthritis. Lancet, 10.1016/
S0140-6736(16)30173-8.
2. Birnbaum H, et al. (2010) Societal cost of rheumatoid arthritis patients in the US. Curr
Med Res Opin 26(1):77–90.
3. Helmick CG, et al.; National Arthritis Data Workgroup (2008) Estimates of the prevalenc eof
arthritis and other rheumatic conditions in the United States. Part I. Arthritis Rheum 58(1):
15–25.
4. Tak PP, Kalden JR (2011) Advances in rheumatology: New targeted therapeutics.
Arthritis Res Ther 13(Suppl 1):S1–S5.
5. McInnes IB, Schett G (2011) The pathogenesis of rheumatoid arthritis. N Engl J Med
365(23):2205–2219.
6. Chorus AM, Miedema HS, Boonen A, Van Der Linden S (2003) Quality of life and work
in patients with rheumatoid arthritis and ankylosing spondylitis of working age. Ann
Rheum Dis 62(12):1178–1184.
7. Andersson U, Tracey KJ (2012) Neural reflexes in inflammation and immunity. J Exp
Med 209(6):1057–1068.
8. Andersson U, Tracey KJ (2012) Reflex principles of immunological homeostasis. Annu
Rev Immunol 30:313–335.
9. Rosas-Ballina M, et al. (2011) Acetylcholine-synthesizing T cells relay neural signals in a
vagus nerve circuit. Science 334(6052):98–101.
10. Waldburger JM, Boyle DL, Pavlov VA, Tracey KJ, Firestein GS (2008) Acetylcholine
regulation of synoviocyte cytokine expression by the alpha7 nicotinic receptor.
Arthritis Rheum 58(11):3439–3449.
11. van Maanen MA, et al. (2009) The alpha7 nicotinic acetylcholine receptor on fibroblast-like
synoviocytes and in synovial tissue from rheumatoid arthritis patients: A possible role for a
key neurotransmitter in synovial inflammation. Arthritis Rheum 60(5):1272–1281.
12. Lu B, et al. (2014) α7 nicotinic acetylcholine receptor signaling inhibits inflammasome
activation by preventing mitochondrial DNA release. Mol Med 20:350–358.
13. Rosas-Ballina M, et al. (2009) The selective alpha7 agonist GTS-21 attenuates cytokine
production in human whole blood and human monocytes activated by ligands for
TLR2, TLR3, TLR4, TLR9, and RAGE. Mol Med 15(7-8):195–202.
14. Borovikova LV, et al. (2000) Vagus nerve stimulation attenuates the systemic inflammatory
response to endotoxin. Nature 405(6785):458–462.
15. Huston JM, et al. (2006) Splenectomy inactivates the cholinergic antiinflammatory
pathway during lethal endotoxemia and polymicrobial sepsis. J Exp Med 203(7):
1623–1628.
16. Meregnani J, et al. (2011) Anti-inflammatory effect of vagus nerve stimulation in a rat
model of inflammatory bowel disease. Auton Neurosci 160(1-2):82–89.
17. van Maanen MA, et al. ( 2009) Stimulation of nicotinic acetylcholine receptors attenuates
collagen-induced arthritis in mice. Arthritis Rheum 60(1):114–122.
18. van Maanen MA, Stoof SP, Larosa GJ, Vervoordeldonk MJ, Tak PP (2010) Role of the
cholinergic nervous system in rheumatoid arthritis: Aggravation of arthritis in nicotinic
acetylcholine receptor α7 subunit gene knockout mice. Ann Rheum Dis 69(9):1717–1723.
19. Levine YA, et al. (2014) Neurostimulation of the cholinergic anti-inflammatory
pathway ameliorates disease in rat collagen-induced arthritis. PLoS One 9(8):e104530.
20. van Maanen MA, Vervoordeldonk MJ, Tak PP (2009) The cholinergic anti-inflammatory
pathway: Towards innovative treatment of rheumatoid arthritis. Nat Rev Rheumatol 5(4):
229–232.
21. Beekwilder JP, Beems T (2010) Overview of the clinical applications of vagus nerve
stimulation. J Clin Neurophysiol 27(2):130–138.
22. Ben-Menachem E (2001) Vagus nerve stimulation, side effects, and long-term safety.
J Clin Neurophysiol 18(5):415–418.
23. Huston JM, et al. (2007) Transcutaneous vagus nerve stimulation reduces serum high
mobility group box 1 levels and improves survival in murine sepsis. Crit Care Med
35(12):2762–2768.
24. Fransen J, van Riel PL (2005) The Disease Activity Score and the EULAR response criteria. Clin
Exp Rheumatol 23(5, Suppl 39):S93–S99.
25. Fransen J, Stucki G, van Riel PLCM (2003) Rheumatoid arthritis measures: Disease
Activity Score (DAS), Disease Activity Score-28 (DAS28), Rapid Assessment of Disease
Activity in Rheumatology (RADAR), and Rheumatoid Arthritis Disease Activity Index
(RADAI). Arthritis Care Res 49(S5):S214–S224.
26. Levine YA, Koopman F, Faltys M, Zitnik R, Tak P-P (2014) Using traditional preclinical
models to guide development of an untraditional inflammation therapy: neurostimulation
of the cholinergic anti-inflammatory pathway in rheumatoid arthritis and inflammatory
bowel disease. Bioelectronic Medicine 1:34–43.
27. Olofsson PS, Levine YA, et al. (2015) Single-pulse and unidirectional electrical activation of
the cervical vagus nerve reduces tumor necrosis factor in endotoxemia. Bioelectronic
Medicine 2:37–42.
28. Choi IY, Gerlag DM, Holzinger D, Roth J, Tak PP (2014) From synovial tissue to peripheral
blood: Myeloid related protein 8/14 is a sensitive biomarker for effective treatment in early
drug development in patients with rheumatoid arthritis. PLoS One 9(8):e106253.
29. Gerlag DM, et al. (2004) Effects of oral prednisolone on biomarkers in synovial tissue
and clinical improvement in rheumatoid arthritis. Arthritis Rheum 50(12):3783–3791.
30. Bonaz B, et al. (2016) Chronic vagus nerve stimulation in Crohn’s disease: A 6-month
follow-up pilot study. Neurogastroenterol Motil 28(6):948–953.
31. Koopman FA, et al. (2016) Autonomic dysfunction precedes development of rheu-
matoid arthritis: A prospective cohort study. EBioMedicine 6:231–237.
32. Tracey KJ (2015) Shock medicine. Sci Am 312(3):28–35.
33. Bruchfeld A, et al. (2010) Whole blood cytokine attenuation by cholinergic agonists
ex vivo and relationship to vagus nerve activity in rheumatoid arthritis. J Intern Med
268(1):94–101.
34. Aletaha D, et al. (2010) 2010 Rheumatoid arthritis classification criteria: An American Col-
lege of Rheumatology/European League Against Rheumatism collaborative initiative.
Arthritis Rheum 62(9):2569–2581.
35. Hochberg MC, et al. (1992) The American College of Rheumatology 1991 revised
criteria for the classification of global functional status in rheumatoid arthritis.
Arthritis Rheum 35(5):498–502.
6of6
|
www.pnas.org/cgi/doi/10.1073/pnas.1605635113 Koopman et al.