Production From Patients With Systemic
Lupus Erythematosus Is Done Through
AIM2 And Not NLRP3
1Center for Translational Medicine, Department of Medical Research, Taichung Veterans General
Hospital, Taichung, Taiwan.
2Division of Allergy, Immunology and Rheumatology, Department of Internal Medicine, Taichung
Veterans General Hospital, Taichung, Taiwan.
3Institute of Biomedical Sciences, National Chung-Hsing University, Taichung, Taiwan.
4Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan.
5Section of Allergy, Immunology and Rheumatology, Department of Internal Medicine, Asia
University Hospital, Taichung, Taiwan.
Jaw-Ji Tsai, MD, PhD, Section of Allergy, Immunology and Rheumatology, Department of Internal Medicine, Asia University
Hospital, Taichung, Taiwan. Tel: +886-4-3706-1688 ext.1878; Fax: +886-4-3706-1673; Email:
Received: June 20, 2017; Accepted: July 17, 2017; Published: November 27, 2017
Citation: SAI T (2017) Dp2-Induced Autoantigen/Autoantibody Production From Patients With Systemic Lupus Erythematosus Is Done Through AIM2 And Not NLRP3. J Rheumatol Arthritic Dis 2(4): 1-6.
Systemic Lupus Erythematosus (SLE) is a disease characterized
by the production of autoantibodies against different autoantigens,
including double stranded DNA (dsDNA). AIM2 (Protein absent in
melanoma 2) is one of the inflammasome proteins which can directly
bind to dsDNA. It is therefore possible to speculate that AIM2 may
contribute to the disease development of SLE. B cell lines obtained
from patients with Dp-allergic SLE were used to investigate the
involvement of AIM2 in autoantibody production. The obtained B
cells were then cultured with Dp2 for inflammasome activation. The
cells-cultured pellet and supernatant were subsequently collected for
the measurement of autoantigens, autoantibodies and inflammatory
cytokines. The inflammasome of AIM2 and NLRP3 was measured
and correlated with autoantibodies production. The B cells were
pretreated with siRNA of AIM2, followed by Dp2 stimulation to
confirm the activation of AIM2. The results showed that Dp2 could
induce inflammasome activation with an increased expression of both
NLRP3 and AIM2, which was associated with the production of TRIM-
21/PGK1, anti-TRIM21/anti-PGK-1 and IL8/IL-1β. In the siRNA AIM2
study, Dp2 induced autoantigen/autoantibody/cytokine production
could be down-regulated by siRNA in association with a decreased
expression of AIM2. Dp2-induced expression of NLRP3 and AIM2
can both be down-regulated by CPP ECP. In Conclusion: Dp2 induced
expression of NLRP3 and AIM2 is associated with autoantigen and
autoantibody production from B cells derived from Dp-allergic SLE.
These autoantigens/autoantibodies can be downregulated only by
siRNA AIM2, and not by NLRP3.
Systemic Lupus Erythematosus (SLE) is an autoimmune
disease characterized by various systemic organ and tissue
damage caused by inflammasome activation. Recently enhanced
inflammasome activity in SLE has been previously reported .
A broad range of microbial, host and environmental triggers
of inflammasome have been reported. There are five receptor
proteins which have been confirmed to assemble inflammasomes,
and include Non-binding Oligomerization Domain (NOD),
Leucine-Rich Repeat (LRR)-containing protein, (NLR) family
members NLRP1, NLRP3 and NLRP4, as well as the proteins
absent in melanoma 2 (AIM2) and pyrin [2, 3].
AIM2 is a new type of inflammasome which is similar to NLR
proteins, and can regulate caspase-1 activation and Interleukin
1 beta (IL-1β) production in response to viral, host and bacterial
dsDNA, and in response to the dsDNA virus vaccinia . AIM2
appears to recognize dsDNA from a variety of microbial species,
including those from mammalian cells.
With the existence of a dedicated cytosolic DNA receptor, AIM2
was postulated on the basis of the observation that both microbial
and host DNA induce caspase 1 activation in an ASC-dependent
manner, but independently of NLRP3, Toll-like Receptors (TLR)
or interferon signaling . A link between AIM2 and several
human diseases has been previously established. An increased
AIM2 expression is associated with psoriasis, abdominal aortic
aneurysm and Systemic Lupus Erythematosus (SLE) [6-8]. In
the case of psoriasis, autoinflammation could be linked to AIM2-
mediated recognition of self-DNA in the cytosol of keratinocytes
. Similarly, AIM2 can facilitate the apoptotic DNA-induced
SLE via arbitrating macrophage functional maturation . By
contrast, reduced AIM2 levels correlate with the development of
prostate and colorectal cancer as it has consistently been shown
that AIM2-deficient mice are hyper-susceptible to colonic cancer
development [11-14]. These reports indicate that in addition to
its role in host defence, AIM2 also plays a major role in tumor
progression, possibly by sensing self-DNA.
Anti-dsDNA is a biomarker of SLE and the concentration is
likely related to the disease activity of SLE. Cytosolic dsDNA can
be binded to AIM2 directly to trigger inflammasome activation.
Its role in IL-1β production, and the link between AIM2 and
related family members of inflammasome in the aetiology of SLE,
renders AIM2 an attractive target for intervention in this disease.
Moreover, chronic arthritis caused by mammalian DNA which
escapes from degradation, has also been shown to be associated
with increased levels of IL-1β and IL-18 [15,16].
Our previous study showed that the Group 2 major allergen
of Dermatophagoides pteronyssinus (Dp2) can induce
inflammasome activation through autoantigen/
Autoantibody production by B cells, and that this autoantigen/
autoantibody production was not down regulated by siRNA
NLRP3 [17,18]. It will be of importance to investigate other family
members of inflammasome, particularly AIM2. In this study both
NLRP3 and AIM2 were measured in the B cell culture, and siRNA
AIM2 was added to the cell to clarify the linkage between AIM2
and autoantigen/autoantibody production in SLE.
Materials and Methods
Selection of Patients
HDM-sensitive SLE patients (mite-specific IgE positive in
their serum as measured by using the Pharmacia CAP System,
Uppsala, Sweden) were selected from the clinic in the Division
of Allergy, Immunology and Rheumatology of Taichung Veterans
General Hospital. Diagnosis of SLE was made according to the
1997 America College of Rheumatology revised classification
criteria for SLE. This study was supported by Taichung Veterans
General Hospital (IRB TCVGH No. CE14019A and CE16110B).
For the purpose of cell separation and culture, 16-ml blood
samples were collected, and the PBMCs were separated by
density centrifugation using the Ficoll-Paque Plus density
gradient (Pharmacia Biotech, Freiburg, Germany) . Purified
B cells from the PBMCs were prepared as previously described
. The B cell preparations were shown to be 95% positive for
the CD19 marker as determined by FACS analysis. The cells were
maintained in a RPMI-1640 medium containing inactivated FBS
(10%), along with 1% streptomycin/penicillin in a humidified
5% CO2 atmosphere.
CPP ECP (NYRWRCKNQN, 1381Da) was synthesized at Angene
Biotech Co., Ltd., Taiwan, and its purity (>90%) was assessed
through analytical high-performance liquid chromatography.
Peptide sequences were confirmed using matrix-assisted laser
desorption/ionization, time-of-flight mass spectrometry at
Angene Biotech Co., Ltd., Taiwan.
Dp 2 Preparation
Purified recombinant protein Dp 2 (RP-DP2C-1) was
purchased from Indoor Biotechnologies (Charlottesville, Virginia,
Enzyme-Linked Immunosorbent Assay (ELISA)
Commercially available ELISA kits were used to determine
both cytokine and autoantibody levels in the cell culture
supernatants. The cell supernatants were collected after Dp2 or
CPP ECP treatment during the indicated times, and the levels of
IL-1β’IL-8’IL-6’ anti-PGK-1 and anti-TRIM-21 were quantified by
ELISA. Plates were read on a SpectraMax M2 microplate reader
(Molecular Devices, CA, USA), and analyzed with SoftMax analysis
software (Molecular Devices, CA, USA). The means of triplicate
ELISA values for each of the dose relationships among these
protein expressions were calculated through the use of linear
Whole cell lysates were prepared as previously described
. After blocking, the blots were incubated with antibodies for
and β-actin, (Cell Signaling, Massachusetts, USA; Santa Cruz
Biotechnology; Millipore, Massachusetts, USA) in TBS overnight
at 4°C, using 0.1% Tween 20, followed by three 10-minute
washes in TBS with 0.1% Tween 20. The membranes were then
incubated using horseradish peroxidase-conjugated, secondary
antibodies (Millipore, Massachusetts and USA) for one hour.
Detection was performed with ECL (Millipore, Massachusetts,
USA), and chemiluminescence was detected by LAS 3000. Band
intensity was analyzed by Multi Gauge software V 3.0.
Statistical analyses were performed using graphpad Prism
5 (graphpad Software, San Diego, CA, USA). Data is presented as
mean ± Standard Error of the Mean (SEM). P-values ≤ 0.05 were
considered statistically significant. The pair student’s t-test was
used to determine the statistical differences between the groups.
Dp2-Induced Cytosolic Protein Expression from B Cells
Dp2 induced cytosolic receptor protein production of
AIM2, NLRP3 and MD2 from B cells were investigated. B cells
derived from patients with Dp-allergic SLE were incubated with
Dp2, followed by the measuring of cytosolic receptor protein
expression. The results showed that all three cytosolic protein
expressions had increased after Dp2 stimulation. Similar results
were obtained through LPS stimulation [Figure 1].
Figure 1: Effect of Dp2-induced cytosolic protein expression from the B cell line.
B cells derived from patients with Dp-allergic SLE (n=4) were incubated with Dp2 for 5 days in a CO2 incubator. Cell pellets were collected for the AIM2, NLRP3 and MD2 measurement. *p< 0.05 in comparison with buffer control.
Effect of SiRNA AIM2 on the Dp2-Induced Autoantigen
Production from B Cells
The B cells derived from patients diagnosed with Dp-allergic
SLE were pre-treated with or without siRNA AIM2, followed by
the stimulation of Dp2. Cell pellets were collected for obtaining
the measurements of autoantigens. The results showed that AIM2
expression could be down regulated through the use of siRNA
AIM2 (n=4). Dp2 induced autoantigen production of both PGK-1
and TRIM-21 from the B cells could be down-regulated by siRNA
AIM2, with no effect on the expression on TIM and Enolase-1 in
comparison with buffer control [Figure 2].
Effect of SiRNA AIM2 on the Dp2-Induced Autoantibody
and Cytokine Production from B Cells
B cells derived from patients with Dp-allergic SLE were
incubated with or without siRNA AIM2, followed by the
stimulation of Dp2. Cell cultured supernatant was collected for
obtaining the measurements of autoantibodies and cytokines.
The results showed that Dp2-induced production of anti-PGK-1
and anti-TRIM21 could be down regulated by siRNA AIM2 (n=4).
Similar results showed that Dp2-induced production of IL-1β and
IL-8 could be down regulated by siRNA AIM2 [Figure 3].
Effect of CPP ECP on Dp2-Induced Cytosolic Protein
Expression from the B cell Line
B cells derived from patients with Dp-allergic SLE were
incubated with Dp2 with or without CPP ECP. Cell pellets were
collected for obtaining the measurements of AIM2, NLRP3 and
MD2. The results showed that CPP ECP could down regulate the
expression of AIM2 and NLRP3, but not MD2 (n=4) [Table 1].
B cells derived from patients with Dp-allergic SLE (n=4) were
incubated with Dp2 with or without CPPecp for 5 days in a CO2
incubator. Cell pellets were collected for the AIM2, NLRP3 and
MD2 measurement. *p< 0.05 in comparison with buffer control.
#p< 0.05 in comparison with Dp2.
Table 1: Effect of CPPecp on Dp2-induced cytosolic protein expression
from the B cell line.
SLE is a disease characterized by the production of
autoantibodies against different autoantigens. Some of these
autoantibodies have exhibited a link between titers and disease
activity, such as the anti-dsDNA autoantibody. More than 95 % of
untreated SLE anti-dsDNA could possibly be detected in the sera.
It is believed that SLE disease activity can be triggered by various
environmental factors including sex hormones, ultraviolet
light, infections and certain medications and chemicals. Our
previous reports have shown that Dp can trigger autoantigen/
autoantibody production from B cells derived from patients with
Dp allergic SLE through bystandard activation [15,16]. There are
five receptor proteins which have been confirmed to assemble
in inflammasome, including NLRPs and AIM2. In this study both
NLRP3 and AIM2 could be up-regulated by Dp2 stimulation,
suggesting that both inflammasome proteins can be induced by
Figure 2: Effect of siRNA AIM2 on the Dp2-induced autoantigen production from B cells.
B cells (n=4) derived from patients with Dp-allergic SLE were pre-treated with or without siRNA AIM2 for 3 days, followed by the stimulation of Dp2
for 5 days in the CO2 incubator. Cell pellets were collected for the measurement of autoantigen. *p< 0.05 in comparison with buffer control. #p< 0.05
in comparison with Dp2
Figure 3: Effect of siRNA AIM2 on the Dp2-induced autoantibody and cytokine production from B cells.
B cells (n=4) derived from Dp-allergic SLE were incubated with or without siRNA AIM2 for 3 days, followed by the stimulation of Dp2 for 5 days in the
CO2 incubator. Cell cultured supernatant was collected for the measurement of autoantibodies and cytokines.
Since AIM2 is a sensor of cytoplasmic DNA, it can bind ds-
DNA directly and is considered to be relevant to the regulation of
autoimmune response in SLE. In this Dp2-induced autoimmune
response, the assembling of AIM2 could be inhibited through
the pretreatment of its siRNA, while the decreased assembling
of AIM2 has been correlated with the down-regulation of
autoantigen/autoantibody production. These results suggest that
the activation and assembling of AIM2 may play at least in part, a
role in the pathogenesis of autoimmune in SLE. The results show
that both AIM2 and NLRP3 could be down-regulated by CPP ECP.
These results suggest that CPP ECP is non-specific in immuno
modulation with regards to the Dp2-induced immune response.
A previous study has shown that Dp2 can induce autoantigen/
autoantibody production in Dp-allergic SLE patients, but our
patient’s autoantigen/autoantibody production was not downregulated
due to the pre-treatment of siRNA NLRP3 . In the
present study, Dp2-induced inflammation activation to generate
autoantigens/autoantibodies is associated with the assembling of
NLRP3 and AIM2 proteins. However, only AIM2 assembling is for
autoantigen/autoantibody production, since this autoantigen/
autoantibody production can be down-regulated by the successful
inhibition of AIM2 through siRNA AIM2. These results suggest
that a biomarker for AIM2 is specific autoantigen/autoantibody
production. The major limitation of this study was that there were
too few patients enrolled, and no figures on decrease in activity
were collected to correlate with the expression of AIM2. This
cannot be clarified until more cases are included. Whether AIM2
can be used as a biomarker for SLE disease activity still requires
further investigation. In conclusion, Dp2-induced autoantigen/
autoantibody production from B cells derived from Dp-sensitive
SLE patients occurs through AIM2, and not NLRP3.
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