Introduction
Hand-foot-and-mouth disease (HFMD) is an acute
infectious disease with high incidence in preschool children,
caused by a variety of enterovirus infections, and the clinical
symptoms include fever, rash and sores on a plurality of sites of
the hands, feet and mouth (1).
Enterovirus 71 (EV71) and Coxsackie virus A16 are common
etiological agents of HFMD, among which, EV71 can cause severe
central nervous system damage and multiple-organ dysfunction and
even become life threatening (2).
EV71 infection can cause immune system dysfunction; T lymphocyte
subsets can secrete a variety of inflammatory cytokines, which can
worsen the disease symptoms (3).
Toll-like receptors (TLRs) are important pattern
recognition molecules; those associated with the viral infection
include TLR2, 3, 4, 7, 8 and 9 (4,5). After
recognizing the virus, TLRs can activate multiple signaling
pathways including the downstream MyD88-dependent and the
MyD88-independent pathways, which regulate the expressions of a
variety of inflammatory cytokines and are involved in inflammation
and immune responses (6).
The present study was designed to analyze the immune
mechanisms of intestinal TLRs and the MAPK signaling pathway after
severe HFMD EV71 infection.
Subjects and methods
Subject information
A total of 86 children admitted to the Xuzhou
Children's Hospital (Jiangsu, China) from January 2013 to January
2016 with a throat-swab-pathologic-examination-confirmed diagnosis
of EV71 HFMD for the first time were contiguously enrolled in the
study. A total of 49 children had a mild disease and 27 children a
severe form of the disease. The mild disease group included 29 boys
and 20 girls, with a mean age of 4.5±0.6 years; the severe disease
group included 17 boys and 10 girls with a mean age of 4.9±0.8
years. In addition, 30 healthy children were enrolled in a control
group, including 18 boys and 12 girls with a mean age of 4.8±0.7
years. Comparisons of general information between the three groups
yielded no significant differences.
Study methods
Under fasting conditions, 6 ml elbow venous blood
samples were collected from each patient, and 2 ml was used for
separating peripheral blood mononuclear cells (PBMC) and testing
for TLRs, 2 ml for immune cytokine testing, and 2 ml for MAPK
signaling pathways testing.
Quantification of TLR and MAPK
signaling molecule mRNA expression
Lymphocyte separation medium was added to peripheral
blood samples and these were then centrifuged. The suspended PBMC
were isolated in Eppendorf tubes. For RNA extraction, 1 ml TRIzol
lysis buffer (Invitrogen Life Technologies, Carlsbad, CA, USA) was
added, and the closed tubes were inverted until well mixed,
chloroform and isopropanol were then added before centrifugation.
The pellets of RNA were washed twice with ethanol; and after the
last concentration, purity and integrity were tested. Good quality
RNA samples were inversely transcribed into cDNA with first-strand
cDNA synthesis kit (Promega Corp., Madison, WI, USA), the
experiments were carried out according to the fluorescence
quantitative PCR kit instructions. Specific fragments from TLR2, 3,
4, 7, 8 and 9 as well as ERK, JNK and p38 mRNA were amplified, mRNA
levels of each target gene were calculated normalizing against
GAPDH RNA levels (internal reference).
Testing of cytokine levels in the
serum
Peripheral blood samples were centrifuged at 2,000 ×
g for 20 min, the upper serum layer was used in an enzyme-linked
immunosorbent assay (ELISA). Briefly, an ELISA kit was used to test
the tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin
(IL)-6 and IL-10 levels in the serum samples, the kit was purchased
from the Sigma (St. Louis, MO, USA), and a microplate reader was
purchased from Beijing Liuyi Instrument Factory (Beijing, China),
and carried out according to the manufacturer's instructions.
Statistical analysis
The SPSS 20.0 software (IBM SPSS, Armonk, NY, USA)
was used for data entry and processing. Measurement data were
expressed as mean ± standard deviation, comparisons among groups
were analyzed with a one-way ANOVA method, and pairwise comparisons
were tested with an LSD-t-test method. Countable data were
expressed as rates and the comparisons among groups were analyzed
with a χ2 test. Finally, a Pearson correlation analysis
of measurement data was done after a normality test. P<0.05 was
considered to indicate a statistically significant difference.
Results
Comparison of the expression levels of
TLR mRNAs among the groups
The average expression levels of TLR3, TLR4, TLR7
and TLR8 mRNAs in the isolated PBMCs of subjects in the three
groups were significantly different (P<0.05), and those of TLR2
and TLR9 mRNAs were not (P>0.05). The average expression levels
of TLR3, TLR4, TLR7 and TLR8 mRNAs in PBMCs of the severe disease
group were significantly higher than those in the other two groups,
with the lowest values being found in the control group (P<0.05;
Table I).
 | Table I.Comparison of the expression levels of
TLR mRNAs in PBMCs of subjects in the three groups. |
Table I.
Comparison of the expression levels of
TLR mRNAs in PBMCs of subjects in the three groups.
| Groups | TLR2 | TLR3 | TLR4 | TLR7 | TLR8 | TLR9 |
|---|
| Control | 1.00±0.16 | 1.00±0.13 | 1.00±0.09 | 1.00±0.19 | 1.00±0.15 | 1.00±0.11 |
| Mild disease | 1.05±0.14 |
1.89±0.26a |
1.91±0.24a |
1.77±0.20a |
1.58±0.23a | 1.08±0.15 |
| Severe disease | 1.09±0.12 |
3.32±0.48a,b |
3.88±0.59a,b |
3.27±0.41a,b |
3.06±0.52a,b | 0.92±0.12 |
Comparison of expression levels of
cytokines in the serum
The average expression levels of TNF-α, IFN-γ, IL-6
and IL-10 in the serum of subjects in the three groups were
significantly different. Average expression levels of TNF-α, IFN-γ,
IL-6 and IL-10 in the serum of the severe disease group were higher
than those in the other two groups, the lowest values were found in
the control group (P<0.05; Table
II).
| Table II.Comparison of expression levels of
cytokines in serum of subjects in the three groups. |
Table II.
Comparison of expression levels of
cytokines in serum of subjects in the three groups.
| Groups | TNF-α, ng/ml | IFN-γ, pg/ml | IL-6, ng/ml | IL-10, pg/ml |
|---|
| Control | 11.48±1.86 |
7.68±0.92 |
23.58±5.86 |
9.14±0.15 |
| Mild disease |
18.59±2.49a |
18.51±2.64a |
47.64±7.12a |
17.79±2.52a |
| Severe disease |
55.75±8.64a,b |
30.24±5.42a,b |
142.86±25.38a,b |
28.58±4.82a,b |
Correlation between TLR levels in
PBMCs and cytokine levels in serum
Pearson correlation analysis showed that the TLR3,
TLR4, TLR7 and TLR8 mRNA levels in PBMCs were positively correlated
with TNF-α, IFN-γ, IL-6 and IL-10 levels in the serum (P<0.05;
Table III).
| Table III.Correlation between TLR mRNA levels in
PBMCs and cytokine levels in serum. |
Table III.
Correlation between TLR mRNA levels in
PBMCs and cytokine levels in serum.
|
| TLR3 | TLR4 | TLR7 | TLR8 |
|---|
|
|
|
|
|
|
|---|
|
| t-test | P-value | t-test | P-value | t-test | P-value | t-test | P-value |
|---|
| TNF-α | 0.284 | 0.035 | 0.301 | 0.030 | 0.346 | 0.023 | 0.348 | 0.022 |
| IFN-γ | 0.315 | 0.032 | 0.281 | 0.026 | 0.348 | 0.022 | 0.310 | 0.024 |
| IL-6 | 0.302 | 0.033 | 0.268 | 0.028 | 0.328 | 0.025 | 0.367 | 0.018 |
| IL-10 | 0.325 | 0.030 | 0.296 | 0.025 | 0.303 | 0.028 | 0.349 | 0.015 |
Comparison of expression levels of
MAPK signaling molecules in PBMCs
The average expression levels of ERK, JNK and p38
mRNA in PBMCs of subjects in the three groups were significantly
different (P<0.05). Those of ERK, JNK and p38 mRNA in PBMCs of
the children in the severe disease group were significantly higher
than those in the other two groups, the lowest values were found in
the control group (P<0.05; Table
IV).
| Table IV.Comparison of expression levels of
MAPK signaling molecules in PBMCs of subjects in the three
groups. |
Table IV.
Comparison of expression levels of
MAPK signaling molecules in PBMCs of subjects in the three
groups.
| Groups | ERK | JNK | p38 |
|---|
| Control | 1.00±0.18 | 1.00±0.14 | 1.00±0.17 |
| Mild disease |
1.83±0.25a |
1.59±0.21a |
1.77±0.27a |
| Severe disease |
3.52±0.58a,b |
3.28±0.52a,b |
2.89±0.39a,b |
Correlation between TLR in PBMCs and
MAPK mRNA levels
Pearson correlation analysis showed that the TLR3,
TLR4, TLR7 and TLR8 mRNA levels in PBMCs were positively correlated
with ERK, JNK and p38 mRNA levels (P<0.05; Table V).
| Table V.Correlation between TLR mRNA levels in
PBMCs and MAPK levels. |
Table V.
Correlation between TLR mRNA levels in
PBMCs and MAPK levels.
|
| TLR3 | TLR4 | TLR7 | TLR8 |
|---|
|
|
|
|
|
|
|---|
|
| t-test | P-value | t-test | P-value | t-test | P-value | t-test | P-value |
|---|
| ERK | 0.485 | 0.016 | 0.512 | 0.014 | 0.482 | 0.016 | 0.509 | 0.016 |
| JNK | 0.483 | 0.017 | 0.472 | 0.018 | 0.518 | 0.013 | 0.542 | 0.012 |
| p38 | 0.527 | 0.012 | 0.542 | 0.010 | 0.509 | 0.015 | 0.567 | 0.009 |
Discussion
The immune function of organisms are activated after
EV71 infection and secreted cytokine levels by a variety of immune
cells increase. On the one hand, these changes are involved in the
organism immune protection; on the other hand they can also cause
immunopathological injury to the organism. The balance of Th1/Th2
cells is an important mechanism regulating immune function. Th1
cells can synthesize TNF-α, IFN-γ and IL-6, which mainly mediate
the cell immune response and play a major role in the antiviral
process (7). Th2 cells can
synthesize IL-10, inhibit excessive activation of Th1 cells,
promote the proliferation of B cells and mainly mediate the humoral
immune response (8). Studies have
confirmed that the functions of Th1 and Th2 cells in children with
HFMD are significantly altered and the synthesis and secretion of
cytokines are increased (9,10). In our study, it was found that the
expression levels of TNF-α, IFN-γ, IL-6 and IL-10 in the serum of
children in the severe disease group were significantly higher than
those in the mild disease and the control groups. This is
consistent with the cited previous study results. The above
cytokines synthesized by Th1 and Th2 cells are not only involved in
the organism's protective immune response, they simultaneously
promote an inflammatory reaction that can cause adverse reactions.
In particular, cytokines synthesized by Th1 cells can cause a
systemic inflammatory response syndrome (11) when attempting to clear the virus
infections, that is why a Th2 cell response antagonizes the
excessive inflammatory response in vivo, acting as the
organism's own compensatory defense mechanism (12). When the cytokines synthesized by Th1
and Th2 in pediatric patients with HFMD are increased
significantly, they cause a strong traumatic response syndrome
(13).
Currently, the changes in levels of Th1 and Th2
secretions in pediatric patients with HFMD, and the mechanisms
involved in the regulation of the resulting inflammatory cytokine
syntheses are not clear. TLRs are considered the most important
pattern recognition molecules in vivo, after a viral
infection, a conformational change occurs in them, which leads to
activation of a signal transduction through the MyD88-dependent and
MyD88-independent pathways. An inflammatory response ensues with
diverse cytokines regulating the expression of other inflammatory
factors. The present study found that the expression levels of the
TLR3, TLR4, TLR7 and TLR8 mRNAs in PBMCs of children in the severe
disease group were significantly higher than those in the mild
disease and the control groups. And that those levels were
positively correlated with TNF-α, IFN-γ, IL-6 and IL-10 levels in
the serum. It is therefore possible that TLRs regulate the Th1 and
Th2 cytokine production in the child patients with HFMD and help
regulate the immune response of HFMD (14,15).
Following TLR activation a cascade of events occurs
leading especially to MAPK activation. Then, the signaling pathways
mediated by the three signal molecules ERK, JNK and p38 activate
c-Jun and c-Fos in the nucleus of lymphocytes and start the
transcription of a variety of inflammatory factors (16,17). The
present study found that the expression levels of ERK, JNK and p38
mRNA in PBMCs of pediatric patients, in the severe disease group,
were significantly higher than those in the mild disease group and
the control group, and, that the TLR3, TLR4, TLR7 and TLR8 mRNA
levels in PBMCs were positively correlated with ERK, JNK and p38
mRNA levels. From these results, it looks as though the high
expression levels of TLR3, TLR4, TLR7 and TLR8 in severe EV71 HFMD
regulate cytokine expression by the MAPK pathway and the resulting
exaggerated response causes a severe and dangerous syndrome. It is
possible that an immunological intervention, downregulating the
expression of TLRs, may be a new target in the treatment of severe
HFMD.
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