Discussion
EBV-HLH
is a typical cytokine storm syndrome, and cytokines play a crucial role
in the pathogenesis of EBV-HLH. Wada et
al [8]. observed that EBV-infected patients had
different serum cytokine levels and different severities of clinical
manifestations. The synthesis, secretion, and biological effects of
cytokines are regulated by their genes, and many studies have
demonstrated important roles of cytokine gene polymorphisms in
regulating cytokine levels and influencing the development of
diseases[9-11]. SNPs are the most common form of
gene polymorphisms, and we therefore hypothesized that cytokine SNPs
might play an important role in the pathogenesis of and susceptibility
to EBV-HLH.
Soluble IL-2 receptor (sIL-2R, also known as sCD25) is a marker of
T-lymphocyte activation and is elevated in conditions such as infection,
trauma, malignant hematological disorders, and autoimmune diseases.
sIL-2R is currently used as an indicator of the severity of several
diseases, and its level was shown to be directly proportional to the
degree of inflammation[12]. Excessive activation
of CD8+ T cells and significantly increased serum levels of sIL-2R have
made sIL-2R an important diagnostic and disease marker for HLH in
patients with EBV. The HLH-2004 diagnostic guidelines even added serum
sIL-2R level ≥2400 U/ml as a new diagnostic criterion for
HLH[13]. Furthermore, SNPs of the IL2RAgene encoding sIL-2R are associated with the development of autoimmune
diseases, such as diabetes, multiple sclerosis, and
baldness[14-16].
In the current study, we investigated the three most common IL2RAloci, rs2104286, rs12722489, and rs11594656, and found that the
rs2104286 AA genotype and A allele frequencies were significantly higher
in children with EBV-HLH than in controls, suggesting that these might
be risk factors for the development of EBV-HLH in children. rs2104286 is
located in the first intron of the IL2RA gene, which is not a
coding or regulatory region; however, this SNP has been associated with
several autoimmune diseases, including multiple sclerosis and rheumatoid
arthritis[17]. Carriers of the rs2104286 A allele
have elevated sIL-2R levels, diminished IL-2R signaling, increased
granulocyte-macrophage colony-stimulating factor production by memory
CD4+ T cells, increased frequency of CD25+ naïve T cells, and decreased
CD25 expression on the surface of memory CD127+CD25+ and Treg
cells[15, 18]. Moreover, the rs2104286
polymorphism can affect the activity of enhancer elements in the first
intron of the IL2RA gene and the binding affinity of the
transcription factor TRAF4, as well as mRNA processing and half-life, to
influence IL2RA expression[17]. Dendrouet al. [19] found that the rs2104286 AA
genotype upregulated CD25 (IL-2Rα) expression levels on CD4+ naïve T
cells and CD14+CD16+ monocytes, thereby enhancing T-cell activation, and
this genotypic effect persisted after T cell activation, leading to a
high proportion of CD69+CD4+ naïve T cells that upregulated CD25 in
donors carrying the susceptible rs2104286 allele. We thus deduced that
the rs2104286 polymorphism might affect the pathogenesis of EBV-HLH by
upregulating CD25 expression on CD4+ naïve T cells, thus increasing
sIL-2R levels.
IL-10 is produced by Th2 cells and is an important anti-inflammatory
factor. Cellular secretion of IL-10 is significantly increased during
microbial infections and autoimmune diseases. It is involved in the
development of HLH by modulating immunity and suppressing the
inflammatory response. Significantly elevated serum IL-10 levels are a
characteristic cytokine pattern of HLH[20].
Sánchez et al. [21] reported that certain
SNPs in the promoter region of the IL-10 gene might influence
IL-10 production and response intensity, and were associated with
disease development. IL-10 SNPs were shown to be associated with
various diseases, such as sepsis, asthma, multiple sclerosis, ankylosing
spondylitis, gastric cancer, and coronary heart
disease[4, 21, 22]. In the current study, we
selected rs1800896, rs1800871, and rs1800872 for testing and showed that
the frequencies of the AA genotype and A allele of rs1800896 and the CC
genotype and C allele of rs1800872 were significant higher in children
with EBV-HLH than in controls, suggesting that these might be
predisposing risk factors for the development of EBV-HLH in children.
Wang et al .[9] studied the relationship
between the rs1800872 and children with EBV-HLH, and similarly showed
that the rs1800872 CC genotype and C allele frequencies were higher in
children with EBV-HLH than in patients with infectious mononucleosis
(IM) and in healthy controls. rs1800896 is located at a putative
ETS-like transcription factor-binding site and rs1800872 at a putative
signal transducer and activator of transcription 3-binding site and
regions of negative regulatory function[23, 24].
Polymorphisms at these sites may thus influence transcription factor
binding and consequent IL-10 transcription and IL-10 serum
levels, thus affecting disease susceptibility[25,
26]. IL-10 levels are significantly elevated in children with
EBV-HLH. Numerous studies have shown that the C allele of rs1800872 is
associated with high levels of [27, 28], while the
relationships between the G and A alleles of rs1800896 and IL-10 levels
remain controversial. Rees et al. [24, 29]showed that the rs1800896 A allele enhanced transcription of theIL-10 gene and resulted in high levels of IL-10. In contrast,
Engelhardt et al. [30-32] showed that the
rs1800896 G allele was associated with higher IL-10 production, while
the rs1800896 A allele resulted in lower IL-10 production. The
frequencies of IL-10 polymorphisms differ among
populations[33], and IL-10 plays different roles
in different diseases, although its regulatory mechanisms are not fully
understood. Numerous genetic correlation studies and a few functional
studies have shown that the regulation of IL-10 is complex and
multifactorial. Further studies are therefore needed to determine how
rs1800896 and rs1800872 regulate the production of IL-10 and affect the
susceptibility to and clinical course of EBV-HLH in children. In
addition, the rs1800896 polymorphism can also influence EBV infection
status. Consistent with the results of the present study, Helminenet al. [34] also showed that individuals
carrying the rs1800896 A allele were more susceptible to severe EBV
infection. The rs1800896 and rs1800872 polymorphisms may thus influence
EBV-HLH susceptibility by synergistically regulating IL-10 expression
and EBV infection status.
Haplotype analysis provides more accurate genetic information than
analysis of individual SNPs. Comparisons between children with EBV-HLH
and controls based on estimated haplotype frequencies thus provide a
better understanding of the role of cytokine SNPs in EBV-HLH
susceptibility. The present results showed that the frequency of theIL2RA AGT (rs2104286-rs12722489-rs11594656) haplotype was
significantly increased while that of the GGT haplotype was
significantly decreased in children with EBV-HLH. The frequency of theIL-10 ACC (rs1800896-rs1800871-rs1800872) haplotype was also
significantly increased, whereas the frequencies of the ACA and GTA
haplotypes were significantly decreased. In addition, some of these
haplotypes showed strong linkage disequilibrium, suggesting thatIL2RA and IL-10 haplotype association and linkage
disequilibrium may play important roles in the susceptibility to
EBV-HLH. The results of this study suggest that the IL2RA AGT
haplotype may increase the risk of EBV-HLH; however, no other studies
have simultaneously tested the IL2RA rs2104286, rs12722489, and
rs11594656 loci, and studies of the AGT haplotype in other diseases are
lacking, and further studies are therefore needed to validate the roles
of the AGT and GGT haplotypes. The common IL-10rs1800896-rs1800871-rs1800872 haplotypes are GCC, ACC, and ATA. The ACC
haplotype is associated with moderate IL-10
production[35], but its role in different diseases
is controversial. Marangon et al. [36]showed that this haplotype was a protective factor in diffuse large
B-cell lymphoma, while Gao et al. [28]showed that it was associated with an increased risk of IgA nephropathy.
We also analyzed IFN-γ rs2430561, IRF5 rs2004640, andCCR2 rs2004640, but found no significant associations between
these SNPs and susceptibility to EBV-HLH. IFN-γ is an important cytokine
produced by a variety of immune cells in response to inflammatory
stimuli. It regulates the body’s immune response, and is significantly
elevated in children with EBV-HLH. IFN-γ rs2430561 has been shown
to affect susceptibility to diseases such as systemic lupus
erythematosus, sepsis, ankylosing spondylitis, and breast
cancer[37-40], but no previous association between
this SNP and HLH has been reported. IRF5 is a member of the IRF
transcription factor family that plays important roles in both the
interferon pathway and the Toll-like receptor signaling pathway, and is
closely associated with many autoimmune diseases[41,
42]. IRF5 rs2004640 has been associated with susceptibility to
diseases such as systemic juvenile idiopathic arthritis(sJIA), systemic
lupus erythematosus(SLE), and systemic
sclerosis(SS)[43, 44]. Yanagimachi et
al. [42] investigated the relationship between
rs2004640 and susceptibility to secondary HLH, such as macrophage
activation syndrome and EBV-HLH, and found that the GT/TT genotype at
this locus increased overall susceptibility to secondary HLH, but not to
EBV-HLH, consistent with the results of the present study. The chemokine
receptor CCR2 plays a key role in several diseases, and the CCR2rs1799864 gene polymorphism has also been associated with susceptibility
to diseases such as ischemic stroke and multiple
sclerosis[45, 46]. Ou et
al .[47] found no significant difference in the
rs1799864 polymorphism between children with and without HLH, suggesting
that this locus may be independent of HLH susceptibility, consistent
with the current study. However, the paucity of relevant studies and the
limited numbers of cases mean that further studies are needed to confirm
the relationships between the above genetic polymorphisms and EBV-HLH.
This study had some limitations. First, the relatively small sample size
may have limited the statistical power, and more multicenter studies
with larger sample sizes are therefore needed to validate our findings.
Second, because of technical limitations in the quantitative analysis of
cytokines before 2014, cytokine profiles were not available for more
than half of the patients. Unfortunately, we were therefore unable to
analyze the relationships among SNPs, cytokine secretion, and HLH.