Journal Pre-proof MiR-101-3p down-regulates TLR2 expression, leading to reduction in cytokines production by T. pallidum-stimulated macrophages Tao Huang, Jieyi Yang, Jun Zhang, Wujian Ke, Fei Zou, Chengsong Wan, Liuyuan Wang, Xiaohui Zhang, Fangwen Liang, Shuqing Mei, Qiwei Zhang, Zhili Rong, Bin Yang, Heping Zheng PII:
To appear in:
The Journal of Investigative Dermatology
Received Date: 2 February 2019 Revised Date:
6 November 2019
Accepted Date: 2 December 2019
Please cite this article as: Huang T, Yang J, Zhang J, Ke W, Zou F, Wan C, Wang L, Zhang X, Liang F, Mei S, Zhang Q, Rong Z, Yang B, Zheng H, MiR-101-3p down-regulates TLR2 expression, leading to reduction in cytokines production by T. pallidum-stimulated macrophages, The Journal of Investigative Dermatology (2020), doi: https://doi.org/10.1016/j.jid.2019.12.012. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 The Authors. Published by Elsevier, Inc. on behalf of the Society for Investigative Dermatology.
MiR-101-3p down-regulates TLR2 expression, leading to reduction in cytokines
production by T. pallidum-stimulated macrophages
Tao Huang1,6*, Jieyi Yang1*, Jun Zhang1, Wujian Ke1, Fei Zou2, Chengsong Wan3, Liuyuan
Wang1, Xiaohui Zhang1, Fangwen Liang4, Shuqing Mei5, Qiwei Zhang3, Zhili Rong1,6, Bin
Yang1#, and Heping Zheng1#
University, Guangzhou, China.
Department of Microbiology, Southern Medical University, Guangzhou, China.
Department of Dermatology, Yingde Center for Chronic Disease Control, Yingde, China.
Department of Dermatology, Zhuhai Center Chronic Disease Control, Zhuhai, China.
Cancer Research Institute, School of Basic Medical Sciences, Southern Medical University,
ORCiD IDs: T. Huang, 0000-0002-6701-6555; J. Yang, 0000-0001-8747-744X; J. Zhang,
0000-0001-5044-4919; W. Ke, 0000-0003-4800-4302; F. Zou, 0000-0002-1144-7540; C. Wan,
0000-0001-6847-8658; L. Wang, 0000-0001-5390-7608; X. Zhang, 0000-0002-0275-2543; F.
0000-0002-2770-111X; Z. Rong, 0000-0002-1699-7074; H. Zheng, 0000-0002-9846-975X;
B. Yang, 0000-0002-7155-6079.
Research Center, Dermatology Hospital, Southern Medical University, Guangzhou, China. Department of Occupational Health and Occupational Medicine, Southern Medical
Correspondence: B. Yang ([email protected]
, +8613922207231); H. Zheng
Treponema pallidum (Tp) infection-induced immune responses can cause tissue damage.
However, the underlying mechanism by which Tp infection induces immune response is
unclear. Recent studies suggest a regulatory role of microRNAs (miRNAs) in host immunity.
We assessed whether miRNAs also have a regulatory role in immune response to Tp infection
in vitro. Our results showed that miR-101-3p levels were significantly higher in peripheral
blood mononuclear cells of patients with primary syphilis and those in the serofast state,
while TLR2 levels were higher in syphilitic patients than in healthy controls. In vitro,
stimulation of THP-1 cells with Tp increased miR-101-3p expression. Moreover, miR-101-3p
reduced expression levels of TLR2 mRNA and protein in THP-1 cells via binding to the 3
untranslated region of TLR2. Likewise, miR-101-3p inhibited production of inflammatory
cytokines, including IL-1β, IL-6, TNF-α, and IL-12, in Tp-stimulated macrophages. IL-1β
and IL-6 mRNA expression levels were reduced by transfection of macrophages with a
TLR2-specific small interfering RNA. Conversely, overexpression of TLR2 up-regulated
cytokines expression. Patients with secondary syphilis exhibited the highest levels of plasma
IL-6, which were negatively correlated with miR-101-3p. In conclusion, Tp infection
up-regulates miR-101-3p expression, which in turn inhibits the TLR2 signaling pathway,
leading to reduced cytokines production.
Keywords: Treponema pallidum, miR-101-3p, macrophages, TLR2, inflammatory cytokines
Syphilis is one of the most common sexually transmitted diseases globally and is caused by 2
the spirochet Treponema pallidum subsp. pallidum (Tp) (Radolf et al., 2006, Lafond and
Lukehart, 2006). Syphilis affects nearly 6 million people globally and represents a major
public health issue (Korenromp et al., 2017). The natural history of syphilis has four stages:
primary syphilis, secondary syphilis, latent syphilis, and tertiary syphilis (Ho and Lukehart,
2011). Although treatment with appropriate antibiotics can improve syphilis symptoms, the
evaluation of therapeutic response largely relies on serological tests. Patients with a greater
than four-fold reduction in nontreponemal titers are considered as showing a good serological
response, whereas those with less than four-fold changes in nontreponemal titers, currently
accounting for 30–40%, are referred to as “serofast” (Seña et al., 2011). Great variation in
symptoms and severity can occur owing to differences in host immune responses to Tp.
Serological tests cannot distinguish syphilis in the serofast state from latent syphilis because
of the similar manifestations of these two subtypes.
In recent decades, the importance of innate and adaptive immune responses in the
immunopathogenesis of syphilis has been well appreciated. Spirochetal lipoproteins and/or
synthetic lipopeptides can activate macrophages and dendritic cells (DCs) via CD14 and
Toll-like receptor 1 (TLR1)/TLR2-dependent signaling pathways (Norgard et al., 1996,
Sellati et al., 1998). Although Tp lacks lipopolysaccharide (LPS), a pro-inflammatory
constituent of the outer membranes of Gram-negative bacteria (Ulevitch and Tobias, 1999),
abundant lipoproteins are expressed in Tp (Chamberlain et al., 1989, Fraser et al., 1998). Both
spirochetal lipoproteins and their synthetic analogs (lipopeptides) are potent activators of
innate immune cells. These pathogen-associated molecular patterns (PAMPs) can bind to
monocytes/macrophages and DCs, leading to activation of immune cells (Aliprantis et al.,
1999). Microarray analysis has shown that both TLR1 and TLR2, but not TLR6, were
up-regulated in the skin of patients with secondary syphilis (Cruz et al., 2012), suggesting a
crucial role of lipoprotein-TRL1 and TLR2 signaling in immune response to syphilis
MicroRNAs (miRNA) are short non-coding RNAs that can suppress mRNA expression
by targeting the 3
untranslated regions (3
UTRs) of the respective mRNAs. An addition
to their role in regulating infectious immunity, miRNAs could serve as novel molecular
markers. Although several miRNAs, including miR-155, miR-146, and miR-21, are known to
inhibit TLR-mediated responses upon binding of the TLR ligand to its receptor (Sheedy et al.,
2010, Quinn and O'Neill, 2011), whether and how miRNAs regulate immunity in syphilis
infection remains unknown. In the present study, we aimed to explore the signaling pathway
of the immune response to Tp infection, using microarrays to obtain miRNA expression
profiles of peripheral blood mononuclear cells (PBMCs) from syphilis patients. By
quantitative real-time polymerase chain reaction (RT-qPCR), we verified nine differential
miRNAs in PBMCs from syphilis patients at different stages of the disease. These miRNAs
included miR-101-3p, miR-195-5p, and miR-223-5p, which have been shown to be
associated with inflammation. The data also show that miR-101-3p is differentially expressed
between syphilitic patients and healthy individuals. We verified that miR-101-3p was
differentially expressed in CD14+ cells and used a cellular model to show how miR-101-3p
affects expression of macrophage inflammatory cytokines. Interestingly, miR-101-3p
expression of PBMCs was negatively correlated with plasma interleukin 6 (IL-6) levels in 4
Expression of miR-101-3p is associated with syphilis in monocytes
The microarray results showed that miR-101-3p may be associated with syphilis, as described
previously (Figure S1). We first verified the differential expression of miR-101-3p. As shown
in Figure 1, miR-101-3p expression in PBMCs was significantly higher in patients with
syphilis than in healthy controls. Moreover, PBMCs from patients with serofast and primary
syphilis exhibited expression of miR-101-3p. Interestingly, miR-101-3p expression was
significantly higher in the serofast state than in latent syphilis. It is not clear why serofast
state occurs after syphilitic patients treatment. Interestingly, the expression of miR-101-3p in
the serofast state was significantly higher than that in untreated syphilitic patients and
serologically cured (Figure 1b), suggesting the involvement of miR-101-3p in the
development of the serofast state. In order to determine in which cells miR-101-3p was
differentially expressed, we tested expression of miR-101-3p in the main PBMCs (Figure 1d–
g), showing that it had similar differential expression to that of PBMCs only in CD14+ cells.
Gene ontology functional enrichment analysis of miR-101-3p target genes
To further elucidate the role of miR-101-3p in the pathogenesis of syphilis, potential target
genes of miR-101-3p were predicted using bioinformatics algorithms (miRWalk,
DIANA-microT4, miRanda, miRDB, PICTAR2, and TargetScan) with miRWalk 2.0. Of the
42 pathways with the smallest p-values, miR-101-3p was most likely to be associated with 5
Toll-like receptor pathways, which are known to be involved in immune regulation of
pathogen infection (Figure 2). Studies have shown that the TLR2 and TLR4 pathways have
important roles in bacterial infection. However, as Tp lacks the LPS ligand of TLR4, we first
focused on how miR-101-3p regulates the TLR2 pathway. Previous studies have shown that
TLR2 plays an important role in Tp infection. And TLR2 is one of the target genes of
miR-101-3p. Further analysis suggested that miR-101-3p might regulate TLR2, which is
crucial in pathogen recognition and activation of innate immunity.
miR-101-3p down-regulates TLR2 expression during Tp infection
As shown above, expression of miR-101-3p in CD14+ cells varied with stage of syphilis. We
next assessed whether expression levels of TLR2 in CD14+ cells (Figure 3a) also varied with
stage of syphilis. Indeed, expression levels of TLR2 were higher in primary and secondary
syphilis than in either healthy controls or patients in the serofast stage (Figure 3a).
Interestingly, TLR2 expression was significantly higher in serologically cure than serofast
state. And the expression of miR-101-3p is opposite. To further investigate the relationships
between miR-101-3p and TLR2, expression levels of miR-101-3p were assessed in THP-1
cells treated with Tp. As shown in Figure 3b, miR-101-3p expression was significantly
increased 24 h and 48 h after incubation with Tp. Expression of TLR2 was also increased at
24 h, followed by a reduction at 48 h (Figure 3b).
To determine the role of miR-101-3p in TLR2 expression, expression levels of TLR2
were measured in PBMCs and THP-1 cells treated Tp, followed by incubation with either
miR-101-3p mimic or inhibitor. As expected, the miR-101-3p mimic down-regulated 6
expression of TLR2 mRNA and protein (Figure 3c) 48 h after incubation. Conversely, the
miR-101-3p inhibitor up-regulated expression levels of both TLR2 mRNA and protein
(Figure 3c). To assess whether miR-101-3p directly regulated the 3
generated luciferase reporters encoding the normal and a mutated TLR2 3
in Figure 3d, luciferase activity was significantly suppressed by miR-101-3p in comparison
with controls 48 h after transfection. By contrast, luciferase activity was up-regulated by the
miR-101-3p inhibitor. These results demonstrate that miR-101 negatively regulates TLR2
UTR of TLR2, we UTR. As shown
miR-101-3p down-regulates the expression of inflammatory cytokines in macrophages
Yang TC (Lin LR et al., 2018) showed that Tp promotes macrophage polarization and
activates inflammasome pathway. As miR-101-3p down-regulates TLR2 expression of
macrophages, we next explored whether miR-101-3p suppressed expression of inflammatory
cytokines associated with the TLR2 signaling pathway in macrophages. In these experiments,
we processed the cells as in Figure 4a. As expected, treatment of macrophages with Tp
markedly increased expression levels of several pro-inflammatory cytokines, including IL-1β,
IL-6, IL-12, and tumor necrosis factor alpha (TNF-α) (Figure 4b,c). However, Tp-induced
up-regulation of cytokines expression was significantly inhibited by miR-101-3p mimic. By
contrast, miR-101-3p inhibitor augmented Tp-induced up-regulation of cytokines expression.
To determine the involvement of TLR2 in cytokines expression mediated by miR-101-3p,
TLR small interfering RNA (TLR-siRNA) and pcDNA-3.1(-)-TLR2 were used to
down-regulate and up-regulate TLR2, respectively. As shown in Figure 4d, silencing of TLR2 7
greatly attenuated Tp-induced elevations in IL-1β and IL-6 mRNA levels. Conversely,
overexpression of TLR2 increased expression levels of IL-1β and IL-6 mRNA (Figure 4f).
We also used peptidoglycan (PGN), TLR2 agonist, instead of Tp to study the effect of
miR-101-3p on activation of TLR2 pathway (Figure 4e). The data show that PGN can
up-regulate the expression of IL1β, IL6 and TNF-a, but no miR-101-3p (Supplementary
material S2). And PGN-induced up-regulation of cytokines expression was significantly
inhibited by miR-101-3p mimics. By contrast, miR-101-3p inhibitors augmented
PGN-induced up-regulation of cytokines expression.
Expression of inflammatory cytokines in syphilitic patients at different stages
As noted above, expression levels of miR-101-3p varied with the stage of syphilis and
miR-101-3p inhibited Tp-induced production of cytokines in macrophages. Hence, we
assessed whether circulating levels of inflammatory cytokines also varied with the stage of
syphilis. A panel of inflammatory cytokines, including IL-1β, IL-6, IL-12, and TNF-α, in
plasma were measured using an ELISA assay. Plasma levels of inflammatory cytokines,
particularly IL-6 and IL-12, varied greatly with the stage of syphilis. Notably, secondary
syphilis exhibited the highest levels of IL-6, while primary and serologically cured syphilis
displayed the lowest levels. By contrast, expression levels of both TNF- α and IL-1β were
similar at the different stages of syphilis.
After Tp infection, mammalian hosts mount robust humoral and cellular immune responses 8
aimed at spirochetal clearance (Arroll et al., 1999, Leader et al., 2007, Leader et al., 2003,
Salazar et al., 2007). However, Tp can escape the host immune response, resulting in
persistent infection. So far, the mechanisms of immune regulation in Tp infection have not
been clear. Recent studies have indicated that miRNAs participate in the pathogenesis of a
variety of infectious diseases (Benz et al., 2016, Fu et al., 2011, Qi et al., 2012, Wang et al.,
2015), but whether miRNAs regulate syphilis immunity remained unknown. Our previous
study demonstrated that miR
overexpressed in the PBMCs of syphilitic patients in comparison with healthy controls. These
three miRNAs have the potential to regulate inflammation. A target of miR-223 is TLR4, of
which LPS is a ligand. Considering the lack of LPS antigen in Tp, however, we did not
initially focus on miR-233. We had also performed some experiments to study the effects of
miR-195 on macrophage inflammation, but the results were not in line with expectations.
5p, and miR
We demonstrated here that levels of miR-101-3p in PBMCs of syphilitic patients vary
with subtype of syphilis, suggesting a pathogenic role of miR-101-3p in the development of
syphilis. As PBMCs are a mixed group of various cell types, we tested expression of
miR-101-3p in the main PBMC components to determine which of them showed differential
expression of miR-101-3p. Interestingly, we found that miR-101-3p expression was similar to
that of PBMCs only in CD14+ cells.
Previous studies have demonstrated the involvement of miR-101-3p in various diseases.
For example, genomic loss of miR-101 leads to overexpression of histone methyltransferase
EZH2 in cancer (Varambally et al., 2008). Moreover, down-regulation of miR-101-3p is
associated with rheumatic heart disease, an autoimmune disease provoked by S. pyogenes 9
infection (Hai et al., 2015). The present study showed that miR-101-3p inhibited the TLR2
pathway via targeting the TLR2 3
function as major pro-inflammatory agonists during spirochetal infection (Salazar et al.,
2009). TLR2 plays an important part in the inflammatory response in syphilis (Lukehart et al.,
1980). Tp contains abundant lipoproteins, which can activate macrophages and DCs via
CD14 and TLR1- and TLR2-dependent signaling pathways (Lien et al., 1999, Radolf et al.,
1995). Accordingly, transcripts for both TLR1 and TLR2, but not TLR6, are up-regulated in
the skin of patients with secondary syphilis (Cruz et al., 2012). Importantly, we demonstrated
here that TLR2 was highly expressed in PBMCs of syphilitic patients and those who were
serologically cured, in comparison with both healthy controls and patients in the serofast state,
suggesting a regulatory role for TLR2 in the immune response to syphilis.
UTR in syphilis. TLRs are PAMPs and are considered to
The TLR2 signaling pathway regulates the expression of inflammatory cytokines (Kigerl
et al., 2010, Snodgrass et al., 2013), while TpN47 activates the TLR2 pathway and induces
the release of pro-inflammatory cytokines, including IL-1β, IL-6, IL-12, and TNF-α
(Brightbill et al., 1999). These cytokines can activate Th1 cells, resulting in elimination of Tp.
IL-6 appears to be the major cytokines in syphilis immunity, with higher levels of IL-6
observed in secondary syphilis compared with other subtypes of syphilis. Plasma levels of
other cytokines did not differ among the subtypes of syphilis. In the present study, we also
found a link between miR-101-3p and TRL2 in response to Tp infection. Based on these
results, we postulate that miR-101-3p inhibits TRL2 expression, leading to a reduction in
cytokines production (Figure 6), and that miR-101-3p levels in syphilitic patients may
determine the levels of cytokines that result in the development of various subtypes of 10
syphilis. For instance, in syphilitic patients, high levels of miR-101-3p decrease TLR2
expression, resulting in a reduction in cytokines production and leading to the development of
the serofast state. Conversely, low levels of miR-101-3p increase TLR2 expression, resulting
in increased cytokines production and leading to a serological cure. Again, this concept
requires further validation.
In conclusion, miR-101 targets the TLR2 3
UTR, resulting in inhibition of TLR2
expression, with a consequent reduction in cytokines production in Tp infection. miR-101
negatively regulates cytokines levels and, as cytokines contribute to the elimination of Tp, it
is likely that miR-101 levels determine the development of subtypes of syphilis.
Measurement of miR-101 levels could facilitate differentiation between the serofast state and
MATERIALS AND METHODS
A total of 15 healthy controls and 108 syphilitic patients, comprising 56 untreated patients
with acute syphilis (15 primary, 18 secondary, 23 latent) and 52 patients who received
standard treatment (28 in the serofast stage and 24 serologically cured), were enrolled in this
study (details in Table 1).
Collection of samples
Peripheral blood samples were collected from patients at the STD clinic of the Dermatology
Hospital of Southern Medical University. Diagnosis of syphilis was made according to
Chinese syphilis prevention and treatment guidelines (version 2015). Briefly, primary syphilis 11
was considered to manifest as localized ulcers, whereas secondary syphilis was characterized
by generalized skin rashes. Latent syphilis was diagnosed by persistent positive results for a
specific treponemal antibody test without symptoms. Serological cure was defined as a
positive result for a treponemal test using the Tp particle agglutination assay and nonreactive
with a nontreponemal test such as the rapid plasma reagin test. The serofast state refers to a
situation in which nontreponemal antibodies decline (often adequately) after treatment but
fail to completely revert to nonreactive. Persons with low pretreatment titers are also
sometimes said to be serofast when there is minimal ( 4-fold) or no change following
treatment. Blood samples from patients with primary, secondary, and latent syphilis were
collected prior to treatment.
Exclusion criteria included: (1) co-infection with human immunodeficiency virus,
condyloma acuminata, or other sexually transmitted diseases; (2) patients with autoimmune
diseases or those receiving anti-pro-inflammatory or immunosuppressive therapy, or those
treated with antibiotics within the past 6 months.
This study was approved by the Ethics Committee of the Dermatology Hospital of Southern
Medical University. The objectives, procedures, and potential risks were verbally explained
to all participants. Written informed consent was obtained from all participants prior to
inclusion in this study.
The THP1 cell line was maintained in RPMI 1640 medium (HyClone, Logan, UT, USA)
supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/mL
penicillin, and 100 µg/mL streptomycin. All cells were maintained at 37 °C and 5% CO2. For 12
analysis of THP-1 macrophage response to microbial ligands, log-phase cells were seeded at
5×105 cells/mL in a 24-well plate. Cells were stimulated with PMA (0.1 µg/mL)
(Sigma-Aldrich) for 24 h. Tp was obtained from vaccinated rabbit testicles. To study the role
of miR-101-3p in macrophages, a specific mimic and inhibitor were transfected into
macrophages after 24 h incubation with Tp. The supernatants of cell cultures were collected
for cytokines measurements.
Quantitative RT-PCR for analysis of miRNA
PBMCs were isolated from venous blood by a standard procedure of Ficoll gradient
centrifugation, performed according to the manufacturer's instructions. Total RNA was
extracted using TRIzol reagent (Invitrogen Waltham, MA, USA). RNA was quantified using
a NanoDrop One spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and
stored in RNase free water at -80 °C.
RT-qPCR was performed using a Mir-X miRNA RT-qPCR SYBR Kit (Takara
Biomedicals, Shiga, Japan) according to the manufacturer’s instructions. Briefly, each 1 ug of
total RNA was reversely transcribed to cDNA using the Mir-X miRNA First-Strand Synthesis
Kit (Takara Biomedicals, Shiga, Japan). The miRNA expression was analyzed in duplicate
and normalized to U6 using a LightCycler 480 (Roche Applied Science, Basel, Switzerland).
Fold changes in miRNA were calculated using the comparative Ct method.
psiCHECK-2 vector (Promega, Madison, WI). THP-1 cells were transfected with miR-101
mimic/inhibitor or controls, followed by co-transfection with 3
UTR of TLR2, its antisense sequence, and a mutant 3
UTR were cloned into the
UTR-luc or mutant 3
UTR-lucl for the luciferase activity assay. After 48 h incubation, cells were lysed and the
luciferase assay was performed using a dual luciferase assay system (Promega, Madison, WI).
Each experiment was performed in triplicate. The firefly luciferase activity and the Renilla
luciferase activity were measured by the luminometer (Promega, Madison, WI). The relative
luciferase activity is presented by the ratio of the firefly luciferase activity and the Renilla
luciferase activity (internal reference).
The miRNA mimics were small, chemically modified double-stranded RNAs that mimic
endogenous miRNAs and enable miRNA functional analysis. The miRNA inhibitors were
small, chemically modified single-stranded RNA molecules designed to specifically bind to
and inhibit endogenous miRNA molecules and enable miRNA functional analysis by
down-regulation of miRNA activity.
Overexpression and suppression of TLR2
An siRNA targeting TLR2 (siRNA-TLR2) with the sense sequence was synthesized by
RiboBio (Guangzhou, China). A scrambled siRNA from RiboBio was used as a negative
control. For siRNA-mediated inhibition of TLR2 gene expression, THP-1 cells were
transfected with TLR2 siRNA or scrambled siRNA at a final concentration of 50 nM, using
the RNA MAX siRNA Transfection Reagent (Invitrogen) according to the manufacturer’s
instructions. Silencing efficiency was estimated at protein levels by western blotting.
The TLR2 sequence (NCBI accession no. NM_001318787) was synthesized by the
Beijing Genomics Institute Company (China) and cloned into the pcDNA3.1(-) vectors. The
recombinant plasmid pcDNA3.1(-)-TLR2 harboring the correct TLR2 coding sequence and
the control vector pcDNA3.1(-) were transfected into THP-1 cells with lipofectamine LTX 14
(Invitrogen) for 48 h.
Whole-cell protein extracts were boiled in sodium dodecyl sulfate (SDS)/β-mercaptoethanol
sample buffer, separated by SDS-polyacrylamide gel electrophoresis, and then transferred to
PVDF membranes (Amersham Pharmacia Biotech, St. Albans, Herts, UK) by electrophoretic
transfer. Specific proteins were detected by the respective antibodies. Rabbit anti-TLR2
monoclonal antibody was purchased from Abcam (Cambridge, MA, USA), and the mouse
anti-GAPDH monoclonal antibody was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA,
peroxidase-conjugated rabbit anti-mouse or goat anti-rabbit IgG (Cell Signaling Technology,
Danvers, MA, USA), and GAPDH was used as a loading control.
Inflammatory cytokine levels were determined in plasma or the supernatant of THP-1 by
ELISA according to the manufacturer’s instructions with modifications. IL12, TNF-α, IL-6
IL-1β ELISA kits were purchased from ELabscience, UK.
PBMCs were isolated using density centrifugation from peripheral blood with Ficoll-Paque
PLUS (GE Healthcare), and were washed twice in phosphate buffered saline (PBS). Then,
these single cells were labelled with 6 antibodies in 100 µl staining buffer (1% fetal bovine
serum and 0.5M EDTA in PBS), and incubated in the dark at 4°C for 30 minutes. The
antibodies were used as following: mouse anti-human CD3 FITC, mouse anti-human CD4
BV510, mouse anti-human CD8 APC, mouse anti-human CD14 BV421, mouse anti-human
CD11b PE and mouse anti-human CD19 APC-H7. Stained cells were washed twice in 1ml
PBS, and centrifuged at 400 x g for 5 minutes. After washes, these cells were resuspended in
PBS for sorting analysis with FACSAriaIII (BD Biosciences). The sorted CD4+ T cells,
CD8+ T cells, B cells and monocytes from all samples were finally used for miR-101-3p
expression profiles by real-time quantitative polymerase chain reaction (RT-qPCR).
Statistical analyses were performed with the GraphPad Prism software 5.01. Data are
presented as mean ± standard deviation unless otherwise stated. Student’s t-test was used for
comparison between two groups. The correlation analysis was used for IL6 in plasma and
miR-101-3p expression in PBMCs. Significances among multiple groups were determined by
one-way variance with Tukey’s multiple comparison. A p-value less than 0.05 was considered
to indicate a significant difference.
DATA AVAILABILITY STATEMENT
Data that support the findings of this study are available from the corresponding author upon
CONFLICT OF INTEREST
The authors state no conflict of interest.
Ethical approval was obtained from the ethics committee of the Dermatology Hospital of 16
Southern Medical University (Chinese NO.GDDHLS-20170614).
Conceptualization: Heping Zheng and Bin Yang; Data curation: Tao Huang, Jieyi Yang and
Jun Zhang; Resources: Wujian Ke, Fei Zou, Chengsong Wan, LiuYuan Wang, Xiaohui Zhang,
Fangwen Liang and Shuqing Mei; Writing: Tao Huang; Writing - Review and Editing: Tao
Huang Zhili Rong and Qiwei Zhang; Funding Acquisition: Heping Zheng, Bin Yang, Tao
Huang and Jun Zhang; Supervision: Heping Zheng and Bin Yang; Formal Analysis and
Methodology: Tao Huang and Heping Zheng; Project Administration: Heping Zheng and Bin
Heping Zheng, Bin Yang, Fei Zou, Chengsong Wan, Zhili Rong, and Qiwei Zhang
contributed to the conception of the study; Jun Zhang contributed significantly to analysis
and manuscript preparation; Wujian Ke, Xiaohui Zhang, and Liuyuan Wang performed the
data analyses and wrote the manuscript; Fangwen Liang and Shuqing Mei helped perform the
analysis and took part in constructive discussions; and Tao Huang and Jieyi Yang carried out
all the experiments.
This work was supported by the following grants:
1. Guangdong Science and Technology Department 2017A020212008.
2. The National Natural Science Foundation of China (NSFC) 81772240.
3. Medical Science and Technology Foundation of Guangdong Province C2017043.
4. Medical Science and Technology Foundation of Guangdong Province A2016269.
5. Natural Science Foundation of Guangdong Province 2015A030310070.
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Figure 1. Expression of miR-101-3p is associated with syphilis in monocytes. (a,b)
Expression levels of miR-101-3p in PBMCs were assessed by RT-qPCR. a) All stages of
specimens. b) Syphilitic patients mean all untreated patients of primary syphilis, secondary
syphilis, and latent syphilis. These specimens were collected before treatment. Samples from
serofast and serologically cured patients were collected after treatment. (c–f) Expression
levels of miR-101-3p in sorted CD14+, CD4+, CD8+, and CD19+ cells. Normal controls
(n=8), primary syphilis (n=7), secondary syphilis (n=8), latent syphilis (n=8), serofast state
(n=9), and serological cure (n=10).
Figure 2. Gene ontology functional enrichment analysis of miR-101-3p target genes.
DIANA-miRPath v3.0 (htT. pallidum://www.microrna.gr/miRPathv3), an online software
suite dedicated to the assessment of miRNA regulatory roles and the identification of
controlled pathways, was used for this analysis. The x-axis shows the name of the pathway,
the y-axis indicates the count of genes, and colors represent the -log10 of the p-value.
Figure 3. miR-101-3p down-regulates expression of TLR2 during Tp infection. (a) TLR2
was detected in CD14+ cells by flow cytometry. Staining of PE-CD14 and APC-TLR2 in
whole blood after lysis of red blood cells. On the left is a representative of each group; on the
right are statistics for the average fluorescence intensity (mean). Normal controls (n=9),
primary syphilis (n=4), secondary syphilis (n=4), latent syphilis (n=4), serofast state (n=10), 24
and serological cure (n=8) (b) Expression levels of miR-101-3p and TLR2 in THP-1 cells
treated with Tp for 6, 24, and 48 h. (c) PBMCs and THP-1 cells were transfection with mimic
or inhibitor for 24 h, followed incubation with Tp by for 24 h. Left: mRNA levels of TLR2 in
PBMCs was detected by RT-qPCR. Right: mRNA and protein levels of TLR2 in THP-1 cells
was detected by RT-qPCR and western blot, respectively. (d) Luciferase vector with the
TLR2 3'UTR or a mutant sequence was transfected into THP-1 cells. Luciferase activity was
detected after transfection with mimic or inhibitor.
Figure 4. miR-101-3p regulates the expression of inflammatory cytokines in
macrophages. (a) Time points sampled for macrophages that THP-1 was induced by PMA.
(b) (i–v) mRNA levels of IL-1β, IL-6, TNF-α, IL-12A, and IL-12B as detected by RT-qPCR.
(c) (i–vi) Cell supernatants were used to detect the expression of inflammatory cytokines
using ELISA. (d) Above: western blot for TLR2; below: mRNA levels of IL-1β and IL-6 as
detected by RT-qPCR. (e) (i-iii) mRNA levels of IL-1β, IL-6 and TNF-α, as detected by
RT-qPCR. (vi-iv) Cell supernatants were used to detect the expression of inflammatory
cytokines using ELISA. (f) Above: western blot for TLR2; middle: mRNA levels of IL-1β
and IL-6 as detected by RT-qPCR; below: the levels of IL-1β and IL-6 of supernatant as
detected by Elisa.
Figure 5. Expression of inflammatory cytokines in syphilitic patients at different stages
(a) Expression of inflammatory factors in patients at different stages of syphilis. (b) The
correlation analysis was used for IL6 in plasma and miR-101-3p expression in PBMCs. And 25
the plasma and PBMCs come from one individual. Data are shown as mean ± SEM;
**P<0.01, ***P<0.001. (Control n=10, primary n=8, secondary n=13, latent n=15, serofast
n=10, serological cure n=8.)
Figure 6. Role of miR-101-3p in regulating cytokines expression. Tp activates the TLR2
signaling pathway, leading to production of proinflammatory cytokines such as IL-1β and
IL-6, possibly via NF-κB signaling (Manish et al., 2005, Sunhwa et al., 2009, Kigerl et al.,
2010). Tp can also increase the expression levels of miR-101-3p in macrophages, resulting in
a reduction in IL-1β and IL-6 production via inhibition of TLR2.
Table 1. Patient information collected