Molecules 2013, 18, 3841-3858; doi:10.3390/molecules18043841
Phenylmethimazole Suppresses dsRNA-Induced Cytotoxicity
and Inflammatory Cytokines in Murine Pancreatic Beta Cells
and Blocks Viral Acceleration of Type 1 Diabetes in NOD Mice
Kelly D. McCall 1,2,3,4,5,6,*, Martin J. Schmerr 7, Jean R. Thuma 3, Calvin B. L. James 2,3,5,
Maria C. Courreges 1, Fabian Benencia 2,5,6, Ramiro Malgor 2,3,5,6 and Frank L. Schwartz 1,3,6
1 Department of Specialty Medicine, Ohio University Heritage College of Osteopathic Medicine,
Athens, OH 45701, USA
2 Department of Biomedical Sciences, Ohio University Heritage College of Osteopathic Medicine,
Athens, OH 45701, USA
3 The Diabetes Institute, Ohio University Heritage College of Osteopathic Medicine, Athens,
OH 45701, USA
4 Department of Biological Sciences, Ohio University College of Arts & Sciences, Athens,
OH 45701, USA
5 Molecular & Cellular Biology Program, Ohio University College of Arts & Sciences, Athens,
OH 45701, USA
6 Biomedical Engineering Program, Ohio University Russ College of Engineering & Technology,
Athens, OH 45701, USA
7 Department of Natural Sciences, Central Ohio Technical College Newark, OH 43055, USA
* Author to whom correspondence should be addressed; E-Mail: email@example.com;
Tel.: +1-740-593-0926; Fax: +1-740-597-1371.
Received: 16 February 2013; in revised form: 28 February 2013 / Accepted: 22 March 2013 /
Published: 27 March 2013
Abstract: Accumulating evidence supports a role for viruses in the pathogenesis of type 1
diabetes mellitus (T1DM). Activation of dsRNA-sensing pathways by viral dsRNA
induces the production of inflammatory cytokines and chemokines that trigger beta cell
apoptosis, insulitis, and autoimmune-mediated beta cell destruction. This study was
designed to evaluate and describe potential protective effects of phenylmethimazole (C10),
a small molecule which blocks dsRNA-mediated signaling, on preventing dsRNA activation of
beta cell apoptosis and the inflammatory pathways important in the pathogenesis of T1DM.
We first investigated the biological effects of C10, on dsRNA-treated pancreatic beta cells
in culture. Cell viability assays, quantitative real-time PCR, and ELISAs were utilized to
Molecules 2013, 18
evaluate the effects of C10 on dsRNA-induced beta cell cytotoxicity and
cytokine/chemokine production in murine pancreatic beta cells in culture. We found that
C10 significantly impairs dsRNA-induced beta cell cytotoxicity and up-regulation of
cytokines and chemokines involved in the pathogenesis of T1DM, which prompted us to
evaluate C10 effects on viral acceleration of T1DM in NOD mice. C10 significantly
inhibited viral acceleration of T1DM in NOD mice. These findings demonstrate that C10
(1) possesses novel beta cell protective activity which may have potential clinical relevance
in T1DM and (2) may be a useful tool in achieving a better understanding of the role that
dsRNA-mediated responses play in the pathogenesis of T1DM.
Keywords: type 1diabetes; dsRNA; beta cell death; phenylmethimazole (C10)
Accumulating evidence supports a role for viruses in the pathogenesis of type 1 diabetes mellitus
(T1DM) [1–20]. Viral infection of beta cells can initiate disease by direct cell damage, beta cell
toxicity from the acute innate antiviral response, or release/induction of endogenous beta cell self-antigens
triggering autoimmune destruction [11,13,14,21,22]. Double-stranded RNA (dsRNA) is a molecular
intermediate formed during the replication cycle of many viruses. Activation of dsRNA-sensing
pathways such as TLR3 and RIG1 by dsRNA leads to beta cell apoptosis and expression of
pro-inflammatory genes known to be important in the development of T1DM [23,24].
Polyinosinic-polycytidylic acid (pIC) is a synthetic dsRNA that mimics host responses triggered by
viral dsRNA, including direct viral toxicity and antiviral responses that can trigger autoimmune
mechanisms. For example, pIC induces apoptosis of beta cells and impairs beta cell function .
High doses of pIC precipitated diabetes in both diabetes-prone BB (DP-BB) and diabetes-resistant
(DR-BB) rats [25,26] and accelerated the onset of T1DM in non-obese diabetic (NOD) mice . In
addition, pIC has been shown to induce insulitis and accelerate the progression to T1DM in RIP B7.1
mice , and induce insulitis, but not T1DM, in BALB/c mice . Type 1 interferons (specifically,
IFN and IFN), which are strongly induced by pIC, have been associated with the pathogenesis of
T1DM [24,30–33], and their therapeutic use in diseases, such as chronic active hepatitis, has triggered
autoimmune diseases including T1DM . These type 1 interferons have been shown to accelerate
the onset of diabetes in NOD mice, induce insulitis in diabetes resistant strains , and are present in
the beta cells of recent-onset T1DM patients [31,36]. pIC also induces other inflammatory mediators
that exacerbate the autoimmune process, including CXCL10 and major histocompatibility (MHC)
Class I and II molecules [37,38].
We have previously shown that phenylmethimazole (C10), a small molecule derivative of
methimazole, effectively blocks dsRNA induction of co-stimulatory and adhesion molecules,
chemokines, pro-inflammatory cytokines and their receptors, and MHC gene expression in thyrocytes,
pancreatic cancer cells, and melanoma cells [37–39]. More recently, we demonstrated that C10 blocks
dsRNA-induced IRF3 homodimer formation in pancreatic ductal epithelioid carcinoma cells , an
important step in the TLR3 and RIG1 signaling pathways [23,41]. This suggests that C10 may have
Molecules 2013, 18
biological activities that protect pancreatic beta cells from dsRNA-induced beta cell death and
upregulation of many cytokines and chemokines involved in the pathogenesis of T1DM. Thus, these
studies were designed to evaluate and describe potential beneficial biological effects C10 may have on
preventing dsRNA activation of beta cell apoptosis and inflammatory pathways important in the
development of T1DM as well as to evaluate efficacy of C10 to block viral acceleration of T1DM in vivo.
2. Results and Discussion
2.1. C10 Protects Pancreatic Beta Cells from dsRNA-Induced Cytotoxicity
In order to study the effects of C10 on dsRNA-induced beta cell cytotoxicity, we utilized two
mouse pancreatic beta cell lines, TC-6 and NIT-1, in culture. NIT-1 cells are derived from the NOD
mouse , which is genetically prone to spontaneously develop autoimmune-mediated T1DM, whereas
TC-6 cells are derived from transgenic mice that do not spontaneously develop autoimmune-mediated
T1DM [43,44]. Thus, the inflammatory response to dsRNA in NIT-1 cells is expected to be most
representative of the inflammatory response to dsRNA that occurs in vivo in the NOD mouse model,
which triggers an autoimmune-mediated (Pattern A) form of T1DM [20,45,46]. However, the effects
of dsRNA on TC-6 beta cell viability is expected to be most representative of the direct viral
cytotoxicity seen in the Pattern B form of T1DM [20,45,46].
Transfection of TC-6 cells with a low concentration of pIC (1 μg/mL) steadily decreased cellular
viability over a 48-hour period (Figure 1A) while higher concentrations of pIC (10 μg/mL) did not
further increase this cytotoxic effect (Figure 1B). Treatment with C10 significantly diminished the
cytotoxic effects of pIC transfection with either low (Figure 1A) or high concentrations (Figure 1B),
although it was not as protective at the higher concentration of pIC. The solvent used for dissolving
C10 (a solution containing 0.25% DMSO) provided no protection of TC-6 cells transfected with either
concentration of pIC except at the 24-hour time point where it provided only minimal protection
Similar results were obtained in NIT-1 cells. Transfection of NIT-1 cells with pIC (1 μg/mL) also
reduced cell viability in a dose-dependent manner (Figure 1C,D) with higher concentrations (10 μg/mL)
inducing greater cytotoxicity (Figure 1D). NIT-1 cells treated with C10 following pIC transfection
(Figure 1C,D) were protected from the cytotoxic effects of pIC at both concentrations (Figure 1C,D)
similar to that seen with the TC-6 cells, while the solvent had no effect on NIT-1 cell viability.
These data are consistent with previous studies demonstrating the cytotoxic effect of pIC in
pancreatic beta cells  and show for the first time that C10 suppresses the acute induction of beta
cell toxicity in response to dsRNA in both transfected beta cell lines. The cytotoxic effect of
transfection with pIC on the NIT-1 cell line was concentration-dependent, while the TC-6 cell line was
much more sensitive to the pIC treatment because the low dose rapidly induced cytotoxicity. This
result contrasts with the observation of Robbins et al. who reported that higher concentrations of pIC
did not increase cytotoxicity in NIT-1 cells . One explanation for this discrepancy may be that the
longer exposure of cells (48 h) to pIC-liposome complexes in our studies results in the activation of
additional factors involved in programmed cell-death pathways.
Molecules 2013, 18
In sum, C10 suppresses the cytotoxic effects of dsRNA on both transfected beta cell lines, suggesting
that C10 may prevent viral induction of beta cell death seen in both Pattern A and B forms of T1DM.
Figure 1. C10 prevents pIC-induced cytotoxicity in pancreatic beta cells in culture. TC-6
and NIT-1 cells were transfected with 1 mg/mL (A & C) and 10 mg/mL (B & D) of pIC.
TC-6 and NIT-1 cells were either mock transfected (— □ —), transfected with pIC
(… ○ …), or transfected with pIC and treated with either DMSO (solvent)(– – Δ – –) or 0.5 mM
C10 (– · ■ – ·) for the indicated times. At 6, 12, 24, and 48 h post-transfection, the viability
of cells was measured using the Cell Titer-Glo Luminescent Cell Viability Assay.
Molecules 2013, 18
2.2. C10 Blocks dsRNA-Induced Upregulation of TLR3 Expression and Signaling Products in
Pancreatic Beta Cells
It is hypothesized that the pancreatic beta cell itself is an important source of the pro-inflammatory
cytokines that mediate beta cell apoptosis, as well as expression/release of intracellular auto-antigens
propagating the autoimmune-mediated beta cell destruction. Furthermore, dsRNA activation of
dsRNA-sensing pathways, such as TLR3, triggers the production of these pro-inflammatory cytokines
and chemokines in beta cells . Since we have previously shown that C10 is a potent inhibitor
of dsRNA-induction of the same pro-inflammatory cytokines and chemokines involved in the
development of T1DM (CXCL10, IFN, TNF, TLR3, and MHC Class I) in other non-immune cell
types [37–40], we hypothesized that C10 may also be an important and potent inhibitor of dsRNA-induced
production of these cytokines and chemokines in pancreatic beta cells. To test this hypothesis, we
transfected NIT-1 cells with pIC in the presence or absence of C10 and evaluated CXCL10, IFN,
TNF, TLR3, and MHC Class I gene expression. Comparative multiplex real-time PCR was used to
characterize the effects of pIC transfection on the expression of TLR3 and TLR3 signaling products
and to determine the effect of C10 or its vehicle (DMSO) in NIT-1 cells in culture. While after 4 h
there was no induction of TLR3 gene expression by pIC transfection (Figure 2A), however, by 6 h
post-transfection, induction of TLR3 gene expression was evident (Figure 2F). Additionally, while
there was no stimulation of TLR3 by 4 h post-pIC treatment, none-the-less, C10 suppressed TLR3
below basal expression following treatment at this time point (Figure 2A), and prevented pIC
upregulation of TLR3 gene expression at 6 h post pIC treatment (Figure 2F). pIC transfection also
significantly increased CXCL10, IFN, and TNF gene expression while C10 significantly blocked
pIC stimulation of CXCL10, IFN, and TNF expression (Figure 2B–D). Similar to its effect on
TLR3 expression, C10 also significantly reduced basal MHC Class I gene expression (Figure 2E).
Although pIC did not up-regulate MHC Class I expression above basal levels, C10 did significantly
reduce its expression below basal levels, suggesting that C10 may be blocking basally-activated
signaling pathways that control its expression. C10 inhibition of pIC-induced TLR3 expression and
downstream signaling molecules in NIT-1 cells was additionally confirmed by quantitative real-time
PCR arrays and similar findings were also obtained in TC-6 cells (Tables 1–3).
In agreement with our gene expression studies above, we observed a significant increase in
CXCL10 protein levels following pIC transfection and significant inhibition of pIC-induced CXCL10
protein levels with C10 treatment (Figure 3). IFN and CXCL10 are thought to be key players in the
pathogenesis of T1DM. As previously mentioned, type 1 interferons (IFN and IFN) have been
associated with the pathogenesis of type 1 diabetes [24,30–36,48], and their therapeutic use in diseases
such as chronic active hepatitis has triggered autoimmune diseases including type 1 diabetes .
These type 1 interferons accelerate the onset of diabetes in NOD mice , induce insulitis in diabetes
resistant strains , and are present in the beta cells of recent-onset diabetic patients [31,36].
CXCL10 expression in NOD mice is responsible for the targeted migration of autoreactive T-cells into
the pancreatic islets . Furthermore, it has been shown that notably high levels of CXCL10,
followed by CCL2 and CCL5 , are constitutively expressed from pancreatic islets, suggesting that
beta cell production of these chemokines is important in the development of T1DM.
Molecules 2013, 18
Figure 2. C10 suppresses pIC-induced expression of TLR3 and of other genes involved in the
inflammatory response in NIT-1 cells. Quantitative Real Time PCR was used to characterize
the expression of TLR3 and cytokines and chemokines involved in mediating insulits and type
1 diabetes. Total RNA was isolated from NIT-1 cells transfected with 1 μg/mL of pIC using
Lipofectamine 2000 (LF2000) plus DMSO (solvent) or 0.5 mM C10 for 4 h (A–E), or 6 h
(F) and gene expression was evaluated using the murine TLR3, CXCL10, IFN, TNF,
and MHC Class I Gene Expression Assays (Applied Biosystems, Carlsbad, CA). Fold
changes in gene expression were calculated using the ΔΔCt method. Statistical analyses
were performed using one-way ANOVAs followed by Bonferroni and Tukey-Kramer post-hoc
analyses. (A) * Indicates a significant reduction in TLR3 gene expression by C10,
p < 0.00001; (B–D) # Indicates a significant induction of gene expression by pIC,
p < 0.00001, and * indicates a significant reduction in pIC-stimulated gene expression by
C10, p < 0.00001; (E) * indicates a significant reduction in basal MHC Class I expression,
p < 0.001; and in (F) # Indicates a significant induction of TLR3 expression by pIC,
p < 0.05, and * indicates a significant reduction in pIC-stimulated TLR3 expression by
C10, p < 0.05.
Molecules 2013, 18
Table 1. Relative fold changes induced by pIC and inhibition by C10 on TLR signaling array.
Toll-Like Receptor 1
Toll-Like Receptor 2
Toll-Like Receptor 3
Toll-Like Receptor 4
Toll-Like Receptor 6
Toll-Like Receptor 7
Toll-Like Receptor 8
Table 2. Relative fold changes induced by pIC and inhibition by C10 on expression of
genes in a TLR signaling array.
Functional Gene Groupings/Gene Name
TLR adaptor proteins
Heat shock 70kDa protein 1A (Hspa1a)
CCAAT/enhancer binding protein β (Cebpb)
Granulocyte macrophage colony-stimulating
Granulocyte colony-stimulating factor (Csf3)
IL-6 Receptor α (IL-6ra)
Prostaglandin-endoperoxide synthase 2
Tumor necrosis factor, alpha-induced protein
NS = > 5-fold, but not statistically significant p > 0.05.
Molecules 2013, 18
Table 3. Relative fold changes induced by pIC and inhibition by C10 on expression of
inflammatory cytokine gene array.
Functional Gene Groupings/Gene Name TC-6 Cells
Glycoprotein 130 (IL-6st)
Tumor necrosis factor receptor superfamily
Integrin β2 (Itgb)
Other inflammatory genes
Caspase 1 (Casp1)
−62.47 <5 <5 <5 <5
NS = > 5-fold, but not statistically significant p > 0.05.
Molecules 2013, 18
Figure 3. C10 blocks pIC-stimulated CXCL10 protein levels. CXCL10 protein levels were
measured in the cell culture supernatants from NIT-1 cells transfected with 1 μg/mL of pIC
plus DMSO (solvent) or 0.5 mM C10 for 4 h using the mouse CXCL10/IP-10 Quantikine
ELISA kit from R & D Systems. Statistical analysis was performed using a one-way
ANOVA followed by Bonferroni and Tukey-Kramer post-hoc analysis. # Indicates a
significant induction of CXCL10 protein levels by pIC, p < 0.05, and * indicates a
significant reduction in pIC-stimulated CXCL10 protein levels by C10, p < 0.05.
Elevated serum levels of CXCL10 have also been observed in patients with T1DM as well as in
islet-cell autoantibody positive individuals who are at risk for developing T1DM [51,52]. Pathogenic
Th1 cells predominately express CXCR3, the receptor for CXCL10, suggesting that CXCL10
accelerates the autoimmune process by promoting the migration of autoreactive Th1 cells to the
pancreas . In our current studies pIC strongly induced the expression of IFN and CXCL10 in
NIT-1 cells by over 120 fold, while C10 completely suppressed pIC-induced expression of both genes
(IFN and CXCL10). Together, the fact that these genes are critical to the development of type 1
diabetes and that C10 dramatically reduces pIC induction of these genes in beta cells further supports
the idea that C10 may be useful for the prevention of viral-induced type 1 diabetes (both pattern A and
pattern B). Presently, the major barrier to such use is the lack of sensitivity and specificity in
identifying those individuals at risk .
2.3. C10 Attenuates Coxsackievirus B4 (CVB4) Acceleration of T1DM in Non-Obese Diabetic (NOD) Mice
Since C10 significantly blocked dsRNA induction of beta cell death and upregulation of cytokines
and chemokines important in the development of T1DM, we investigated the possibility that C10 may
block viral induction of T1DM in vivo. To test the hypothesis that C10 may be useful to block viral
induction of type 1 diabetes, we utilized the coxsackievirus B4 (CVB4)-induced NOD mouse model of
type 1 diabetes . Acceleration of T1DM in NOD mice by CVB4 can only be achieved once a
critical mass of autoreactive T cells is present in pancreatic islets . If this critical mass of
autoreactive T cells is not present in pancreatic islets prior to infection, then CVB4 infection provides
Molecules 2013, 18
long-term protection from disease expression . Serreze et al. previously established that once NOD
mice have accumulated a naturally occurring critical mass of pancreatic islet autoreactive T cells,
which occurs in most NOD mice by 8 weeks of age, a significant acceleration of T1DM can be
observed following inoculation with CVB4 . Thus, we infected 8 week old NOD mice with CVB4
as previously described  and observed the incidence of diabetes over a 16 week period. For this
experiment, groups of mice (infected or un-infected controls) were treated with a sham injection
(intraperitoneal needle stick only) as a stress control, or were treated once daily with intraperitoneal
injections of DMSO (solvent) or C10 (1 mg/kg). Consistent with previous observations , CVB4
infection of 8 week old NOD mice resulted in a rapid acceleration of diabetes, with 50% of mice
becoming diabetic by 2 weeks post CVB4 infection (10 weeks of age) (Figure 4A, shaded region).
Similar results were observed in DMSO-treated NOD mice infected with CVB4, where we observed
that 38% of mice were diabetic by 2 weeks post CVB4 infection (Figure 4A, shaded region). In contrast,
only 7% of C10-treated NOD mice infected with CVB4 were diabetic by that time (Figure 4A, shaded
region), indicating that C10 effectively reduced CVB4 acceleration of T1DM in NOD mice. NOD
mice not treated with virus but treated daily with sham, DMSO, or C10 injections were also included
in this study, however no animals in any of these groups were diabetic by 10 weeks of age (Figure 4A,
dashed lines). After the initial CVB4-induced spike in diabetes within the first 2 weeks (viral
acceleration phase), we observed a plateau in diabetes development in all virus infected groups which
preceded a subsequent gradual increase in diabetes development (Figure 4A). This plateau effect is in
agreement with previous observations , and reflects what we believe to be the period of prolonged
protection from disease expression mentioned above which is conferred by CVB4 in mice that did not
have a ―critical threshold‖ of insulitis present at the time of infection . The gradual increase in
incidence of diabetes following this plateau is likely due to the genetic predisposition of NOD mice to
spontaneously develop T1DM at this age . The pattern of diabetes development in non-virus-
infected NOD mice indicates the spontaneous development of T1DM that is observed in this
genetically predisposed strain of mice. It is noteworthy that we observed no effect of C10 on the
spontaneous (genetically determined component) development of T1DM in the NOD mouse, which
supports our hypothesis that C10’s protective effects are specific to molecular pathways involved in
the environmental induction/acceleration of disease expression.
It is also noteworthy to mention that we observed no apparent toxic effects in the mice treated with
C10. NOD mice treated with C10 for an extended period of time (16 weeks) show no signs of toxicity
related to the C10 treatment [i.e., C10-treated animals are comparable in weight to control-treated
animals (Figure 4B) and showed no additional signs of distress (i.e., cachexia, poor grooming, absence
of normal dietary patterns)] throughout the duration of the experiment. In addition, we observed no
beta cell toxicity of C10 in any of our cell culture experiments described herein, nor have we seen
toxicity of C10 in any of our previously published in vitro or in vivo studies [37–40,56,57]. Together,
this suggests toxicity associated with C10 is minimal and that C10 is likely to be as well tolerated or
possibly better tolerated than its parent compound methimazole.
Molecules 2013, 18
Figure 4. C10 attenuates CVB4 acceleration of type 1 diabetes in NOD mice without toxicity.
Eight week old female mice were either infected intraperitoneally with 500,000 pfu of
CVB4 or mock infected and/or treated with 1 mg/kg C10, DMSO (solvent control), or
sham injection (stress control) as indicated. Incidence of diabetes and weights of mice in
each group were recorded weekly. (A) Effect of indicated treatment on incidence of
diabetes in NOD mice infected with CVB4 (solid lines) or uninfected (dashed lines).
(B) Effect of indicated treatment on weight of NOD mice.
Molecules 2013, 18
C10 was provided by the Interthyr Corporation (Marietta, OH, USA). A 200 mM stock solution of
C10 was dissolved in dimethylsulfoxide (DMSO) (Sigma, St. Louis, MO, USA), which was further
diluted to 0.5 mM for in vitro experiments in culture medium. pIC was purchased from Sigma
(St. Louis, MO, USA). Edward’s CVB4 was kindly provided by Dr. Roger Loria, Virginia
3.2. Cell Culture and Transfections
NIT-1 and TC-6 beta cell lines were purchased from American Type Culture Collection (ATCC)
(Manassas, VA, USA). NIT-1 cells were cultured in Ham's F12K medium (ATCC) supplemented with
10% heat-inactivated dialyzed fetal bovine growth serum (FBS, Gibco, Carlsbad, CA, USA). TC-6
cells were cultured in Dulbecco’s Modified Eagle Medium (Hyclone, Red Bank, NJ, USA)
supplemented with 15% FBS (Hyclone). Cells were incubated at 37 °C in a humidified atmosphere of
5% CO2. Cells were transfected with pIC using Lipofectamine 2000 (LF2000) (Invitrogen, Carlsbad,
CA, USA). Serum-free media with liposome complexes was replaced at 24 h post-transfection with
appropriate cell culture media for experiments lasting longer than 24 h.
3.3. Cellular Viability Assays
Cells were seeded in triplicate at a density of 2.5 × 104 cells per well. The number of viable cells
was quantified with the Cell Titer-Glo Luminescent Cell Viability Assay (Promega, Madison, WI, USA).
Luminescence was measured with the Lumat LB 9507 luminometer (Berthold Technologies, Oak
Ridge, TN, USA). A standard curve was generated to correlate cell numbers with luminescence activity.
3.4. Quantitative Real Time PCR
Total RNA was isolated (RNeasy Kit, Qiagen, Valencia, CA, USA), treated with DNase (RNase-Free
DNAse Kit, Qiagen), and quantified with the RNA 6000 Nano Chip (Agilent Technologies, Santa
Clara, CA, USA) and the 2100 Bioanalyzer (Agilent Technologies). cDNA was synthesized using
the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA, USA).
Pre-amplification of cDNA was done using the TaqMan PreAmp Master Mix Kit (Applied Biosystems,
Carlsbad, CA, USA). Real-time PCR was performed using murine CXCL10, IFN, TNF, TLR3, and
MHC Class I Gene Expression Assays (Applied Biosystems). Non-preamplified cDNA was used for
CXCL10 real-time PCR, whereas pre-amplified cDNA was used to amplify IFN, TNF, TLR3, and
MHC Class I by real-time PCR. Changes in gene expression were calculated using the ΔΔCt method.
3.5. Quantitative Real Time PCR Arrays
TC-6 and NIT-1 cells were transfected with pIC in the presence or absence of C10 or DMSO
(solvent) for 24 h. Total RNA was isolated (RNeasy MiniElute Cleanup Kit, Qiagen) and quantified
with the RNA 6000 Nano Chip (Agilent Technologies) and the 2100 Bioanalyzer (Agilent
Molecules 2013, 18
Technologies). cDNA was synthesized using the RT² First Strand Kit (SABiosciences, Frederick, MD,
USA). Real-time PCR was performed with the Mouse Toll-Like Receptor Signaling Pathway PCR
Array (SABiosciences) and the Mouse Inflammatory Cytokines and Receptors PCR Array
(SABiosciences) using the Mx3000P QPCR system (Stratagene, La Jolla, CA, USA). Fold changes in
gene expression were calculated using the ΔΔCt method. Statistical analysis was performed using the
two-tailed paired Student’s t-test, and a p-value of less than 0.05 was considered significant.
3.6. CXCL10 ELISA
CXCL10 protein levels were measured in the cell culture supernatants using the mouse
CXCL10/IP-10 Quantikine ELISA kit according to the manufacturer’s instructions (R&D Systems,
Minneapolis, MN, USA).
3.7. NOD Mouse Experiment
Mice were housed in a sterile/pathogen free facility. Seven week old wild-type NOD female mice
were obtained from Jackson Labs (Bar Harbor, ME, USA) and allowed to acclimate for 1 week prior to
initiation of studies. Eight week old NOD mice were infected by intraperitoneal (i/p) injection with
5 × 105 plaque forming units of CVB4 . Indicated animals received daily i/p sham injections and/or
daily injections of DMSO (control solvent) or 1 mg/kg C10 following CVB4 infection. Non-infected
control mice received similar sham, DMSO, or C10 treatments. Weight and blood glucose were measured
weekly. Diabetes was defined as blood glucose values exceeding 240 mg/dL on consecutive days
(at least 24 h apart) and confirmed by urinary glucose excretion (Diastix, Bayer, Pittsburgh, PA, USA).
The results described herein indicate that C10 significantly protects beta cells from dsRNA-induced
cytotoxicity and up-regulation of cytokines involved in beta cell destruction in beta cells in culture.
Moreover, we report the novel finding that C10 protects NOD mice from CVB4-induced acceleration
of T1DM. Together, these results demonstrate that C10: (1) possesses novel beta cell protective
activity from dsRNA induction of apoptosis and inflammation; (2) protects genetically predisposed
NOD mice from CVB4-induced acceleration of T1DM, suggesting that C10 may have potential
clinical relevance for T1DM; and (3) may be a useful tool to glean a greater understanding of the role
that dsRNA-mediated responses play in the pathogenesis of T1DM.
This work was supported in part by a National Institutes of Health, NIDDK grant [grant 1R15
DK081192-01 to KDM], the J.O. Watson Endowed Chair for Diabetes Research, the Ohio University
Heritage College of Osteopathic Medicine, the Ohio University Appalachian Rural Health Institute
Diabetes Research Initiative, and the Interthyr Corporation.
This work would not have been possible without the critical input and guidance from the late
Leonard D. Kohn. Kohn contributed to the original idea for the study and helped in the interpretation
of results and revisions to the manuscript prior to his passing in April 2012.
Molecules 2013, 18
KDM, MJS, and FLS contributed to the original ideas for the studies. KDM conducted all
quantitative real time PCR studies in NIT-1 cells and helped carry out NOD mouse studies. MJS
conducted cell viability and real time PCR array studies. JRT and MCC helped carry out NOD mouse
studies. CBLJ, FB, RM, JRT, and MCC made significant intellectual contributions to the in vivo
studies. KDM supervised all aspects of the work, and was primarily responsible for the writing,
editing, and submission of the manuscript. KDM, MJS, and JRT participated in the preparation of
figures. All authors contributed to discussions of the project, the interpretation of the results, and
revisions to the manuscript.
We would like to thank Alex Campolo, William Mudd, Kevin Albert, Kiet Ma, Elizabeth Smith,
and Arlesia Glaspy for their assistance on this project. We would also like to thank David Serreze
(The Jackson Laboratory) and Mark Atkinson (University of Florida) for their discussions and
expertise provided on the NOD mouse model and Roger Loria (Virginia Commonwealth University)
for his generosity in providing the CVB4 for these studies.
1. Stene, L.C.; Rewers, M. Immunology in the clinic review series; focus on type 1 diabetes and
viruses: The enterovirus link to type 1 diabetes: Critical review of human studies. Clin. Exp. Immunol.
2012, 168, 12–23.
2. Viskari, H.; Knip, M.; Tauriainen, S.; Huhtala, H.; Veijola, R.; Ilonen, J.; Simell, O.;
Surcel, H.M.; Hyoty, H. Maternal enterovirus infection as a risk factor for type 1 diabetes in the
exposed offspring. Diabetes Care 2012, 35, 1328–1332.
3. Jaidane, H.; Sane, F.; Hiar, R.; Goffard, A.; Gharbi, J.; Geenen, V.; Hober, D. Immunology in the
clinic review series; focus on type 1 diabetes and viruses: Enterovirus, Thymus and type 1
diabetes pathogenesis. Clin. Exp. Immunol. 2012, 168, 39–46.
4. Hober, D.; Sane, F.; Jaidane, H.; Riedweg, K.; Goffard, A.; Desailloud, R. Immunology in the
clinic review series; focus on type 1 diabetes and viruses: Role of antibodies enhancing the
infection with Coxsackievirus-B in the pathogenesis of type 1 diabetes. Clin. Exp. Immunol. 2012,
5. Richardson, S.J.; Willcox, A.; Bone, A.J.; Foulis, A.K.; Morgan, N.G. The prevalence of
enteroviral capsid protein vp1 immunostaining in pancreatic islets in human type 1 diabetes.
Diabetologia 2009, 52, 1143–1151.
6. Dotta, F.; Censini, S.; van Halteren, A.G.; Marselli, L.; Masini, M.; Dionisi, S.; Mosca, F.; Boggi, U.;
Muda, A.O.; Del Prato, S.; et al. Coxsackie B4 virus infection of beta cells and natural killer cell
insulitis in recent-onset type 1 diabetic patients. Proc. Natl. Acad. Sci. USA 2007, 104, 5115–5120.
7. Willcox, A.; Richardson, S.J.; Bone, A.J.; Foulis, A.K.; Morgan, N.G. Immunohistochemical
analysis of the relationship between islet cell proliferation and the production of the enteroviral
capsid protein, VP1, in the islets of patients with recent-onset type 1 diabetes. Diabetologia 2011,
8. In't Veld, P. Insulitis in the human endocrine pancreas: Does a viral infection lead to inflammation
and beta cell replication? Diabetologia 2011, 54, 2220–2222.
Molecules 2013, 18
9. Chistiakov, D.A. Interferon induced with helicase C domain 1 (IFIH1) and virus-induced
autoimmunity: A review. Viral Immunol. 2010, 23, 3–15.
10. Ylipaasto, P.; Klingel, K.; Lindberg, A.M.; Otonkoski, T.; Kandolf, R.; Hovi, T.; Roivainen, M.
Enterovirus infection in human pancreatic islet cells, islet tropism in vivo and receptor
involvement in cultured islet beta cells. Diabetologia 2004, 47, 225–239.
11. Jun, H.S.; Yoon, J.W. A new look at viruses in type 1 diabetes. Diabetes Metab. Res. Rev. 2003,
12. Dahlquist, G.; Mustonen, L. Childhood onset diabetes--time trends and climatological factors.
Int. J. Epidemiol. 1994, 23, 1234–1241.
13. Brown, D.W.; Welsh, R.M.; Like, A.A. Infection of peripancreatic lymph nodes but not islets
precedes Kilham rat virus-induced diabetes in BB/Wor rats. J. Virol. 1993, 67, 5873–5878.
14. Guberski, D.L.; Thomas, V.A.; Shek, W.R.; Like, A.A.; Handler, E.S.; Rossini, A.A.;
Wallace, J.E.; Welsh, R.M. Induction of type I diabetes by Kilham's rat virus in diabetes-resistant
BB/Wor rats. Science 1991, 254, 1010–1013.
15. Yoon, J.W.; Kominek, H.I. Role of Coxsackie B Virus in the pathogenesis of diabetes mellitus.
In Microorganisms, Autoimmune Diseases; Rose, N.R., Friedman, H., Eds.; Plenum Press:
New York, NY, USA, 1996; pp. 129–158.
16. Yoon, J.W.; McClintock, P.R.; Onodera, T.; Notkins, A.L. Virus-induced diabetes mellitus.
XVIII. Inhibition by a nondiabetogenic variant of encephalomyocarditis virus. J. Exp. Med. 1980,
17. Yoon, J.W.; Austin, M.; Onodera, T.; Notkins, A.L. Isolation of a virus from the pancreas of a
child with diabetic ketoacidosis. N. Engl. J. Med. 1979, 300, 1173–1179.
18. Menser, M.A.; Forrest, J.M.; Bransby, R.D. Rubella infection and diabetes mellitus. Lancet 1978,
19. Craighead, J.E.; McLane, M.F. Diabetes mellitus: Induction in mice by encephalomyocarditis
virus. Science 1968, 162, 913–914.
20. Richardson, S.J.; Willcox, A.; Bone, A.J.; Morgan, N.G.; Foulis, A.K. Immunopathology of the
human pancreas in type-I diabetes. Semin Immunopathol. 2011, 33, 9–21.
21. Horwitz, M.S.; Ilic, A.; Fine, C.; Rodriguez, E.; Sarvetnick, N. Presented antigen from damaged
pancreatic beta cells activates autoreactive T cells in virus-mediated autoimmune diabetes.
J. Clin. Invest. 2002, 109, 79–87.
22. Liu, D.; Cardozo, A.K.; Darville, M.I.; Eizirik, D.L. Double-stranded RNA cooperates with
interferon-gamma and IL-1 beta to induce both chemokine expression and nuclear factor-kappa
B-dependent apoptosis in pancreatic beta-cells: Potential mechanisms for viral-induced insulitis
and beta-cell death in type 1 diabetes mellitus. Endocrinology 2002, 143, 1225–1234.
23. Garcia, M.; Dogusan, Z.; Moore, F.; Sato, S.; Hartmann, G.; Eizirik, D.L.; Rasschaert, J.
Regulation and function of the cytosolic viral RNA sensor RIG-I in pancreatic beta cells.
Biochim. Biophys. Acta 2009, 1793, 1768–1775.
24. Dogusan, Z.; Garcia, M.; Flamez, D.; Alexopoulou, L.; Goldman, M.; Gysemans, C.; Mathieu, C.;
Libert, C.; Eizirik, D.L.; Rasschaert, J. Double-stranded RNA induces pancreatic beta-cell
apoptosis by activation of the toll-like receptor 3 and interferon regulatory factor 3 pathways.
Diabetes 2008, 57, 1236–1245.
Molecules 2013, 18
25. Ewel, C.H.; Sobel, D.O.; Zeligs, B.J.; Bellanti, J.A. Poly I:C accelerates development of diabetes
mellitus in diabetes-prone BB rat. Diabetes 1992, 41, 1016–1021.
26. Sobel, D.O.; Newsome, J.; Ewel, C.H.; Bellanti, J.A.; Abbassi, V.; Creswell, K.; Blair, O. Poly
I:C induces development of diabetes mellitus in BB rat. Diabetes 1992, 41, 515–520.
27. Serreze, D.V.; Hamaguchi, K.; Leiter, E.H. Immunostimulation circumvents diabetes in NOD/Lt
mice. J. Autoimmun. 1989, 2, 759–776.
28. Wen, L.; Peng, J.; Li, Z.; Wong, F.S. The effect of innate immunity on autoimmune diabetes
and the expression of Toll-like receptors on pancreatic islets. J. Immunol. 2004, 172,
29. Moriyama, H.; Wen, L.; Abiru, N.; Liu, E.; Yu, L.; Miao, D.; Gianani, R.; Wong, F.S.;
Eisenbarth, G.S. Induction and acceleration of insulitis/diabetes in mice with a viral mimic
(polyinosinic-polycytidylic acid) and an insulin self-peptide. Proc. Natl. Acad. Sci. USA 2002, 99,
30. Baldeon, M.E.; Chun, T.; Gaskins, H.R. Interferon-alpha and interferon-gamma differentially
affect pancreatic beta-cell phenotype and function. Am. J. Physiol. 1998, 275, C25–C32.
31. Foulis, A.K.; Farquharson, M.A.; Meager, A. Immunoreactive alpha-interferon in insulin-secreting
beta cells in type 1 diabetes mellitus. Lancet 1987, 2, 1423–1427.
32. Stewart, T.A.; Hultgren, B.; Huang, X.; Pitts-Meek, S.; Hully, J.; MacLachlan, N.J. Induction of
type I diabetes by interferon-alpha in transgenic mice. Science 1993, 260, 1942–1946.
33. Lang, K.S.; Recher, M.; Junt, T.; Navarini, A.A.; Harris, N.L.; Freigang, S.; Odermatt, B.;
Conrad, C.; Ittner, L.M.; Bauer, S.; et al. Toll-like receptor engagement converts T-cell
autoreactivity into overt autoimmune disease. Nat. Med. 2005, 11, 138–145.
34. Fabris, P.; Betterle, C.; Floreani, A.; Greggio, N.A.; de Lazzari, F.; Naccarato, R.; Chiaramonte, M.
Development of type 1 diabetes mellitus during interferon alfa therapy for chronic HCV hepatitis.
Lancet 1992, 340, 548.
35. Alba, A.; Puertas, M.C.; Carrillo, J.; Planas, R.; Ampudia, R.; Pastor, X.; Bosch, F.;
Pujol-Borrell, R.; Verdaguer, J.; Vives-Pi, M. IFN beta accelerates autoimmune type 1 diabetes in
nonobese diabetic mice and breaks the tolerance to beta cells in nondiabetes-prone mice.
J. Immunol. 2004, 173, 6667–6675.
36. Huang, X.; Yuang, J.; Goddard, A.; Foulis, A.; James, R.F.; Lernmark, A.; Pujol-Borrell, R.;
Rabinovitch, A.; Somoza, N.; Stewart, T.A. Interferon expression in the pancreases of patients
with type I diabetes. Diabetes 1995, 44, 658–664.
37. Harii, N.; Lewis, C.; Vasko, V.; McCall, K.; Benavides-Peralta, U.; Sun, X.; Ringel, M.; Saji, M.;
Kohn, L. Thyrocytes express a functional toll-like receptor 3(TLR3): Overexpression can be
induced by viral infection, Reversed by Phenylmethimazole, and is associated with Hashimoto’s
autoimmune thyroiditis. Mol. Endocrinol. 2005, 19, 1231–1250.
38. McCall, K.D.; Harii, N.; Lewis, C.J.; Malgor, R.; Kim, W.B.; Saji, M.; Kohn, A.D.; Moon, R.T.;
Kohn, L.D. High Basal Levels of Functional Toll-Like Receptor 3 (TLR3) and Non-Cannonical
Wnt5a Are Expressed in Papillary Thyroid Cancer (PTC) and Are Coordinately Decreased by
Phenylmethimazole Together with Cell Proliferation and Migration. Endocrinology 2007, 148,
Molecules 2013, 18
39. Schwartz, A.L.; Malgor, R.; Dickerson, E.; Weeraratna, A.T.; Slominski, A.; Wortsman, J.; Harii,
N.; Kohn, A.D.; Moon, R.T.; Schwartz, F.L.; et al. Phenylmethimazole decreases Toll-like
receptor 3 and noncanonical Wnt5a expression in pancreatic cancer and melanoma together with
tumor cell growth and migration. Clin. Cancer Res. 2009, 15, 4114–4122.
40. Courreges, M.C.; Kantake, N.; Goetz, D.J.; Schwartz, F.L.; McCall, K.D. Phenylmethimazole
blocks dsRNA-induced IRF3 nuclear translocation and homodimerization. Molecules 2012, 17,
41. Akira, S.; Takeda, K.; Kaisho, T. Toll-like receptors: Critical proteins linking innate and acquired
immunity. Nat. Immunol. 2001, 2, 675–680.
42. Hamaguchi, K.; Gaskins, H.R.; Leiter, E.H. NIT-1, A pancreatic beta-cell line established from a
transgenic NOD/Lt mouse. Diabetes 1991, 40, 842–849.
43. Efrat, S.; Linde, S.; Kofod, H.; Spector, D.; Delannoy, M.; Grant, S.; Hanahan, D.; Baekkeskov, S.
Beta-cell lines derived from transgenic mice expressing a hybrid insulin gene-oncogene.
Proc. Natl. Acad. Sci. USA 1988, 85, 9037–9041.
44. Poitout, V.; Stout, L.E.; Armstrong, M.B.; Walseth, T.F.; Sorenson, R.L.; Robertson, R.P.
Morphological and functional characterization of beta TC-6 cells—An insulin-secreting cell line
derived from transgenic mice. Diabetes 1995, 44, 306–313.
45. Gianani, R.; Campbell-Thompson, M.; Sarkar, S.A.; Wasserfall, C.; Pugliese, A.; Solis, J.M.;
Kent, S.C.; Hering, B.J.; West, E.; Steck, A.; et al. Dimorphic histopathology of long-standing
childhood-onset diabetes. Diabetologia 2010, 53, 690–698.
46. Rowe, P.A.; Campbell-Thompson, M.L.; Schatz, D.A.; Atkinson, M.A. The pancreas in human
type 1 diabetes. Semin. Immunopathol. 2011, 33, 29–43.
47. Robbins, M.A.; Maksumova, L.; Pocock, E.; Chantler, J.K. Nuclear factor-kappaB translocation
mediates double-stranded ribonucleic acid-induced NIT-1 beta-cell apoptosis and up-regulates
caspase-12 and tumor necrosis factor receptor-associated ligand (TRAIL). Endocrinology 2003,
48. Pelegrin, M.; Devedjian, J.C.; Costa, C.; Visa, J.; Solanes, G.; Pujol, A.; Asins, G.; Valera, A.;
Bosch, F. Evidence from transgenic mice that interferon-beta may be involved in the onset of
diabetes mellitus. J. Biol. Chem. 1998, 273, 12332–12340.
49. Frigerio, S.; Junt, T.; Lu, B.; Gerard, C.; Zumsteg, U.; Hollander, G.A.; Piali, L. Beta cells are
responsible for CXCR3-mediated T-cell infiltration in insulitis. Nat. Med. 2002, 8, 1414–1420.
50. Giarratana, N.; Penna, G.; Amuchastegui, S.; Mariani, R.; Daniel, K.C.; Adorini, L. A vitamin D
analog down-regulates proinflammatory chemokine production by pancreatic islets inhibiting T
cell recruitment and type 1 diabetes development. J. Immunol. 2004, 173, 2280–2287.
51. Shimada, A.; Morimoto, J.; Kodama, K.; Suzuki, R.; Oikawa, Y.; Funae, O.; Kasuga, A.;
Saruta, T.; Narumi, S. Elevated serum IP-10 levels observed in type 1 diabetes. Diabetes Care
2001, 24, 510–515.
52. Nicoletti, F.; Conget, I.; Di Mauro, M.; Di Marco, R.; Mazzarino, M.C.; Bendtzen, K.; Messina, A.;
Gomis, R. Serum concentrations of the interferon-gamma-inducible chemokine IP-10/CXCL10 are
augmented in both newly diagnosed Type I diabetes mellitus patients and subjects at risk of
developing the disease. Diabetologia 2002, 45, 1107–1110.
Molecules 2013, 18 Download full-text
53. Rhode, A.; Pauza, M.E.; Barral, A.M.; Rodrigo, E.; Oldstone, M.B.; von Herrath, M.G.; Christen, U.
Islet-specific expression of CXCL10 causes spontaneous islet infiltration and accelerates diabetes
development. J. Immunol. 2005, 175, 3516–3524.
54. Rewers, M. Challenges in diagnosing type 1 diabetes in different populations. Diabetes Metab. J.
2012, 36, 90–97.
55. Serreze, D.V.; Ottendorfer, E.W.; Ellis, T.M.; Gauntt, C.J.; Atkinson, M.A. Acceleration of type 1
diabetes by a coxsackievirus infection requires a preexisting critical mass of autoreactive T-cells
in pancreatic islets. Diabetes 2000, 49, 708–711.
56. Benavides, U.; Gonzalez-Murguiondo, M.; Harii, N.; Lewis, C.J.; Schwartz, A.L.; Giuliani, C.;
Napolitano, G.; Dagia, N.M.; Malgor, R.; McCall, K.D.; et al. Phenylmethimazole inhibits
production of proinflammatory mediators and is protective in an experimental model of endotoxic
shock*. Crit. Care. Med. 2011, 40, 886–894.
57. McCall, K.D.; Holliday, D.; Dickerson, E.; Wallace, B.; Schwartz, A.L.; Schwartz, C.; Lewis,
C.J.; Kohn, L.D.; Schwartz, F.L. Phenylmethimazole blocks palmitate-mediated induction of
inflammatory cytokine pathways in 3T3L1 adipocytes and RAW 264.7 macrophages.
J. Endocrinol. 2010, 207, 343–353.
58. Webb, S.R.; Loria, R.M.; Madge, G.E.; Kibrick, S. Susceptibility of mice to group B coxsackie
virus is influenced by the diabetic gene. J. Exp. Med. 1976, 143, 1239–1248.
Sample Availability: Samples of the compound (C10) are available from the Interthyr Corporation.
© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license