Production from Patients with
Systemic Lupus Erythematosus and Its
Modulation by Cppecp
Chi-Xin YE1, Sheng-Jie YU1, Ching-Yun CHANG1, Jaw-Ji TSAI1, 2, 3, 4*
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.
Jaw-Ji Tsai, MD, PhD, Division of Allergy, Immunology and Rheumatology, Taichung Veterans General Hospital, 1650 Taiwan Boulevard Section 4, Taichung 40705, Taiwan, Phone: +886-4-2359-2525 ext:3013; Fax:+886-4-2359-2705; E-mail:
Received: December 27, 2016; Accepted: March 3, 2017; Published: April 4, 2017
Citation: Jaw-Ji TSAI, Chi-Xin YE, Sheng-Jie YU, Ching-Yun CHANG (2017) Dp2-Induce Autoantigen/Autoantibody Production from Patients- with Systemic Lupus Erythematosus and Its Modulation by Cppecp. J Rheumatol Arthritic Dis 2(1):1-6.
Epidemiology studies have shown that the prevalence of allergies
in patients with Systemic Lupus Erythematosus (SLE) was higher than
in those of normal, healthy subjects. Although many patients with SLE
also have allergies, the immunological events triggering the onset and
progression of the clinical manifestations of SLE by allergens have yet
to be clarified. Our previous report showed that three autoantigens,
Phosphoglycerate Kinase 1 (PGK-1), Triosephosphate Isomerase
(TIM) and enolase were identified through the use of autologous
serum in B cell lysate. This was derived from House Dust Mites (HDM)
allergic SLE patients after Dp2 stimulation, where the concentration of
anti-PGK-1 was significantly up-regulated after Dp2 stimulation, when
compared to HDM allergic without SLE patients and healthy subjects.
In this study, we further investigated Dp2 stimulated autoantigens
(PGK-1 and TRIM-21) and autoantibodies (anti-PGK-1 and TRIM-21)
production and their relationship with inflammasomal activation IL-
1β production. Both Peripheral Blood Mononuclear Cell (PBMC) and B
cell lines derived from patients with Dermatopgagoides pteronyssinus
(Dp)-allergic SLE was included in this study. CPPecp and siRNA
knockdown were added to evaluate their effect on cell activation.
Our results showed that Dp2 could up-regulate PGK-1,TRIM -21,anti-
PGK-1 and anti-TRIM-21 in B cell lines, and also up-regulate enolase
and PGK-1 in PBMC. The expression of IL-1β and NLRP3 was also
increased through Dp2 stimulation. Although both autoantigens and
autoantibodies were upregulated, the increment of autoantigen and
autoantibodies were abolished after being co-cultured with CPPecp. In
the siRNA NLRP3 study only the increment of TRIM-21 and anti-PGK-1
was abolished. In conclusion, our study demonstrated that allergen
exposure in the patients with HDM-sensitive SLE may play a role in
the incremental expression of autoantigens and autoantibodies. In
patients with Dp allergic SLE, Dp2 could upregulate the production
of auto-antigen and auto-antibodies, while contributing to the proinflammatory
cytokine secretion and disease activation. Dp2-induced
auto-antigen and auto-antibody production could be abolished by
being co-cultured with Dp2 and CPPecp, indicating that CPPecp has
the potential to be a new immune-modulatory agent for the treatment
of autoimmune diseases in the future.
Systemic Lupus Erythematosus (SLE) is a multifactorial
systemic autoimmune disorder which affects multiple organs
including the skin, lungs, kidneys, and heart . In some SLE
patients, autoantibodies may target specific proteins in the cytosol
or nucleus. These proteins are called autoantigens. Since these
self-antigens are normally covered from the extracellular space
by both the nuclear membrane and the cell membrane, the major
issue is how these self-antigens are exposed or upregulated, and
then detected by the immune system.
In recent years, bystander activation of the immune
system in SLE patients has been related to environment
factors. Bystander activation means there is an indirect or nonspecific
activation of autoimmune cells which is caused by the
inflammatory environment present during infection . This can
lead to an enhanced processing and presentation of self antigens,
which induces the expansion or spreading of the immune
response towards different self-antigens [3, 4]. House Dust Mites
(HDM) is the most commonly found aeroallergens worldwide.
Dust mite allergens similar to the environmental pathogen,
Lipopolysaccharide (LPS), can combine with Toll Like Receptors
(TLR) to cause an inflammatory cell-mediate immune response.
Its major allergen, Dermatophagoides pteronyssinus 2 (Dp2),
can internalize to epithelium and augment toll-like receptormediated
proinflammatory signaling with IL-6 and IL-8 secretion
. Dp2 is able to both trigger human B lymphocyte activation,
and induce B cell proliferation. Dp2 can induce expressionof IL-
1β, TNF-α and IL-8, and up-regulate MD2, TLR4 induction in B
cells, which enhance the host’s immune response . Dp2 can
induce bystander activation of B cells derived from patients with
SLE . Whether SLE patients with hypersensitivity to Dp2 were
more susceptible to inflammasome activation and thus prone to
autoantibody production remains unclarified.
In recent years, the correlation of inflammasome
activation of NLRP3 and disease activity of SLE has been reported.
Kahlenberg, et al demonstrated an upregulation of NLRP3 and
caspase-1 in lupus nephritis biopsies . Furthermore, immune
complexes such as complement and autoantigen/autoantibody
complex have been demonstrated to induce inflammasome
activation through the stimulation of TLR dependent activation
of NFκB, which then causes the NLRP3 inflammasome activation
[9, 10]. Neutrophil extracellular traps have been recently
demonstrated, and could activate caspase-1, causing IL-1β and
IL-18 over expression in macrophages derived from SLE patients
. The importance of Dp2 in inflammasomal activation has also
been demonstrated by our group . Dp2 can induce expression
of NLRP3, ASC, Caspase-1, IL-1β and IL-6 activity in THP-1
cells. The pro-inflammatory cytokines IL-1β and IL-6 were also
upregulated in the Peripheral Blood Mononuclear Cells (PBMCs)
derived from HDM allergic patients after Dp2 stimulation. The
Dp2-induced inflammasomal activation can be down regulated
by cell penetration peptides derived from human eosinophil
cationic proteins (CPPecp) .
The aim of this study was to investigate whether Dp2
could induce inflammasomal activation with autoantigen/
autoantibody production in SLE, and to also determine whether
it could be modulated by CPPecp.
Materials and Methods
Selection of patients
HDM-sensitive SLE and asthma 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 the National Science
Council, Republic of China (MOST 104-2314-B-075A-015- ) and
Taichung Veterans General Hospital (IRB TCVGH No. CE14019
For 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 .
B cell preparations were 95% positive for the CD19 marker as
determined by FACS analysis. The cells were maintained in a
RPMI-1640 medium containing 10% heat inactivated FBS and
1% streptomycin/penicillin in a humidified 5% CO2 atmosphere.
CPPecp (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 with
matrix-assisted laser desorption/ionization, time-of-flight mass
spectrometry at Angene Biotech Co., Ltd., Taiwan.
Dp 2 preparation
Purified recombinant protein Dp2 (RP-DP2C-1) was
purchased from Indoor Biotechnologies (Charlottesville, Virginia,
Enzyme-linked Immunosorbent Assay (ELISA)
Commercially available ELISA kits were used to
determine cytokine and autoantibody levels in the cell culture
supernatants. The cell supernatants were collected after Dp2 or
CPPecp treatment during the indicated times, and the levels of IL-
1β,IL-8,IL-6,IL-17,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 by linear regression.
Whole cell lysates were prepared as previously
described . After blocking, the blots were incubated with
antibodies for anti-human NLRP3,ASC,PGK-1,Enolase-1,TRIM-
21,MD-2 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 with 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
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
Effect of CPPecp on Dp2-induced autoantigen and
autoantibody production from PBMCs
PBMCs derived from patients with Dp-allergic SLE (n=6)
were stimulated with Dp2 in conjuction with or without CPPecp,
followed by the autoantigen (Figure 1A) and autoantibody (Figure
1B) measurements. The results showed that both TRIM-21 and
Enolase-1 had increased after Dp2 stimulation. The increments
of both TRIM-21 and Enolase-1 were diminished after Dp2 coculture
with CPPecp. When the anti-PGK-1 and anti-TRIM-21
were measured, they showed to have a trivial effect onDp2 regarding production of the two autoantibodies.
production of the two autoantibodies.
Figure 1:Effect of CPPecp on Dp2-induced autoantigen and autoantibody production from PBMCs. PBMCs derived from patients with Dp-allergic SLE (n=6) were incubated with Dp2 in conjunction with or without CPPecp for 5 days in a CO2 incubator. Cell pellets were collected for the autoantigen determination (A) and supernatant for the autoantibody measurement (B). *p< 0.05 in comparison with buffer control.
Effect of CPPecp on Dp2-induced autoantigen and
autoantibody production from B cell lines
Figure 2:Effect of CPPecp on Dp2-induced autoantigen and autoantibody
production from the B cell line. 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
autoantigen determination (A) and supernatant for the autoantibody
measurement (B). Cell pellets were collected for the NLRP3 and MD2
measurement (C). *p< 0.05 in comparison with buffer control. #p< 0.05
in comparison with Dp2.
B cell lines derived from patients with Dp-allergic
SLE (n=4) were incubated using Dp2 in conjunction with or
without CPPecp, followed by the autoantigen and autoantibody
measurements. The results showed that both PGK-1 and TRIM-
21 had significantly increased after Dp2 stimulation.
The increment of autoantigen was decreased after Dp2 coculture
with CPPecp (Figure 2A). When the anti-PGK-1 and anti-
TRIM-21 were measured, there were also significant increases
in both of their autoantibody production after Dp2 stimulation.
However, the increment of anti-PGK-1 was decreased after Dp2
co-culture with CPPecp (Figure 2B). When both the NLRP3 and
MD2 were measured, there showed only a significant increase of
NLRP3 production, but not MD2. The increment of NLRP3 was
also decreased after Dp2 co-cultured with CPPecp (Figure 2C).
Effect of CPPecp on Dp2-induced on IL-1β production
from PBMCs and B cell lines
PBMCs (n=6, Figure 3A) and B cells (n=4, Figure 3C)
derived from patients with Dp-allergic SLE and Dp-allergic
rhinitis (n=9, Figure 3B) were incubated with Dp2 in conjunction
with or without CPPecp for 5 days. The results showed that IL-
1β increased after Dp2 stimulation in both groups of Dp-allergic
patients in PBMCs and B cells (Figure. 3). The increment of IL-1β
decreased after Dp2 was co-cultured with CPPecp (Figure. 3).
Effect of siRNA NLRP3 on the Dp2-induced autoantigen
production from B cell lines
B cells derived from patients with Dp-allergic SLE
(n=4), were pre-treated with siRNA NLRP3 followed by the
stimulation of Dp2. The cell pellets were collected for autoantigen
measurement (Figure. 4).
Figure 3:Effect of CPPecp on Dp2-induced on IL-1β production from PBMCs and B cells. PBMCs (n=6, A) and B cells (n=4, C) derived patients with Dp-allergic SLE and PBMCs from Dp-allergic rhinitis (n=9, B) were incubated with Dp2 in conjunction with or without CPPecp for 5 days. Cell supernatant was collected for the measurement of IL-1β. *p< 0.05 in comparison with buffer control. #p< 0.05 in comparison with Dp2.
Figure 4:Effect of siRNA NLRP3 on the Dp2-induced autoantigen
production from B cells. B cells (n=4) derived from patients with Dpallergic
SLE were pre-treated with or without siRNA NLRP3 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.
The results showed that both TRIM-21 and PGK-1 had
increased after Dp2 stimulation. The increment of TRIM-21 had
decreased after pre-treatment with siRNA NLRP3. In the siRNA
knockdown study, the production of NLRP3 was specifically
decreased after being pre-treated with siRNA NLRP3. There was
no effect on the production of ASC after being pre-treated with
Effect of siRNA NLRP3 on the Dp2-induced autoantibody
production from B cell lines
B cells derived from patients with Dp-allergic SLE
were pre-treated with siRNA NLRP3 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 both
anti-PGK-1 and anti-TRIM-21. The results showed there were
significant increases in anti-PGK-1 and anti-TRIM-21 production
through Dp2 stimulation. The Dp2 upregulated increment of anti-
PGK-1 (Figure 5A) production had decreased, but not for anti-
TRIM-21 (Figure 5B) after pre-treatment with siRNA NLRP3.
Although many SLE patients were suffering from HDM
allergies, the immunological events triggering the onset and
progression of the clinical manifestations of SLE by allergens
remains unclarified. In this study, we confirmed that Dp2, a major
allergen of HDM, could induce proinflammatory cytokine IL-
1βproduction with NLRP3 activation, and also demonstrated the
enhanced production of autoantigens and autoantibodies in the
same culture cells derived from patients with Dp-allergic SLE. In
the autoantibody study, Dp2 could up-regulate the expression of
anti-PGK-1 and anti-TRIM-21 production in B cell lines but not
PBMC, indicating that there were only a few antibodies producing
Figure 5:Effect of siRNA NLRP3 on the Dp2-induced autoantibody and IL-1β production from B cells. B cells (n=4) derived from Dp-allergic SLE were incubated with or without siRNA NLRP3 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 (A,B) and IL-1β (C).
B cells in the PBMC. When B cell lines were cultured with Dp2
in conjunction with CPPecp, the increment of anti-PGK-1 was
abolished from being associated with the decreased expression of
NLRP3 and IL-1β. These results indicated that CPPecp abolished
anti-PGK-1 production at least in part from downregulation of
In the autoantigen study, PBMCs could up-regulate
the expression of TRIM-21/enolase through Dp2 stimulation.
However when cells were cultured with Dp2 in conjunction with
CPPecp, the increment of Dp2-stimulated TRIM-21/enolase
could be abolished, with no effect on autoantibody production
in the cultured supernatant. These results indicated that while
more autoantigens (including monocyes and lymphocytes) were
present in PBMCs, there were only limited antibodies producing
B cells in the PBMCs. These limited Dp2-stimulated B cells in
the PBMC may also not be producing significant amounts of
autoantibodies in the culture supernatant. This is unlikely due to
the B cells in the PBMCs being unaffected by CPPecp, since CPPecp
could down-regulate HDM allergen-induced proinflammatory
cytokine production, by inhibiting IL-1β expression in the same
cell culture supernatant.
In our previous study, CPPecp was shown to downregulate
Dp2-induced inflammasome activation, through the
production of pro-inflammatory cytokine IL-1β and IL-6 in PBMCs
derived from patients with Dp allergic asthma . Similar
results obtained in this study showed that CPPecp could down
regulate Dp2-stimulated IL-1β production from both PBMCs and
B cells derived from Dp-allergic SLE. The Dp2 stimulated NLRP3
expression from B cells could be also down-regulated by CPPecp.
Since CPPecp could down-regulate inflammasome
activation, it could also abolish the increment of TRIM-21 and
enolase expression in PBMCs, along with the increment of
TRIM-21 and PGK-1 in B cell lines after Dp2 co-cultured with
CPPecp. These results indicate that CPPecp down-regulated
Dp2-induced autoantigen production could be handled through
the modulation of inflammation activation. When the NLRP3
in B cells was silenced with siRNA, despite NLRP3 and IL-1β
expression possibly being inhibited, there was only a trivial effect
on the Dp2-induced autoantigen and autoantibody production.
These results suggest that the expression of autoantigen and
autoantibodies by B cells are not simply modulated by NLRP3.
Since it has been reported, there have been many members in the
family of NOD-Like Receptors (NLR) involved in the sensing of
pathogens . When molecular mechanisms are involved in the
sensing of Dp2 during inflammasomal activation, other cytosolic
innate immunity receptors could also be involved, in addition to
NLRP3, in the production of autoantigens and autoantibodies.
In summary, the environmental allergen Dp may play
a role in the pathogenesis of autoimmune disease through the
activation of inflammasome in B cells, and also enhance the
expression of autoantibodies. Modulation of inflammasome
activation caused by Dp2 may be of importance towards the
prevention of the development of autoimmune disease. Both
autoantigens and autoantibodies can be downregulated by
CPPecp though upregulating NLRP3, although siRNA NLRP3
can only partially inhibit their expression. A detailed pathway
towards inflammasome activation by Dp2 remains unclarified
until all members of the NOD-Like Receptor (NLR) family have
been investigated in any future studies.
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