MiR-101-3p down-regulates TLR2 expression, leading to reduction in cytokines production by T. pallidum-stimulated macrophages

MiR-101-3p down-regulates TLR2 expression, leading to reduction in cytokines production by T. pallidum-stimulated macrophages

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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:

S0022-202X(20)30001-4

DOI:

https://doi.org/10.1016/j.jid.2019.12.012

Reference:

JID 2246

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.

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MiR-101-3p down-regulates TLR2 expression, leading to reduction in cytokines

2

production by T. pallidum-stimulated macrophages

3

Tao Huang1,6*, Jieyi Yang1*, Jun Zhang1, Wujian Ke1, Fei Zou2, Chengsong Wan3, Liuyuan

4

Wang1, Xiaohui Zhang1, Fangwen Liang4, Shuqing Mei5, Qiwei Zhang3, Zhili Rong1,6, Bin

5

Yang1#, and Heping Zheng1#

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1

7

2

8

University, Guangzhou, China.

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3

Department of Microbiology, Southern Medical University, Guangzhou, China.

10

4

Department of Dermatology, Yingde Center for Chronic Disease Control, Yingde, China.

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5

Department of Dermatology, Zhuhai Center Chronic Disease Control, Zhuhai, China.

12

6

Cancer Research Institute, School of Basic Medical Sciences, Southern Medical University,

13

Guangzhou, China.

14

#

15

([email protected], +8613725109560).

16

ORCiD IDs: T. Huang, 0000-0002-6701-6555; J. Yang, 0000-0001-8747-744X; J. Zhang,

17

0000-0001-5044-4919; W. Ke, 0000-0003-4800-4302; F. Zou, 0000-0002-1144-7540; C. Wan,

18

0000-0001-6847-8658; L. Wang, 0000-0001-5390-7608; X. Zhang, 0000-0002-0275-2543; F.

19

Liang,

20

0000-0002-2770-111X; Z. Rong, 0000-0002-1699-7074; H. Zheng, 0000-0002-9846-975X;

21

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

0000-0001-9969-5727;

S.

Mei,

1

0000-0002-4364-7336;

Q.

Zhang,

22

ABSTRACT

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Treponema pallidum (Tp) infection-induced immune responses can cause tissue damage.

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However, the underlying mechanism by which Tp infection induces immune response is

25

unclear. Recent studies suggest a regulatory role of microRNAs (miRNAs) in host immunity.

26

We assessed whether miRNAs also have a regulatory role in immune response to Tp infection

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in vitro. Our results showed that miR-101-3p levels were significantly higher in peripheral

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blood mononuclear cells of patients with primary syphilis and those in the serofast state,

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while TLR2 levels were higher in syphilitic patients than in healthy controls. In vitro,

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stimulation of THP-1 cells with Tp increased miR-101-3p expression. Moreover, miR-101-3p

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reduced expression levels of TLR2 mRNA and protein in THP-1 cells via binding to the 3

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untranslated region of TLR2. Likewise, miR-101-3p inhibited production of inflammatory

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cytokines, including IL-1β, IL-6, TNF-α, and IL-12, in Tp-stimulated macrophages. IL-1β

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and IL-6 mRNA expression levels were reduced by transfection of macrophages with a

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TLR2-specific small interfering RNA. Conversely, overexpression of TLR2 up-regulated

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cytokines expression. Patients with secondary syphilis exhibited the highest levels of plasma

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IL-6, which were negatively correlated with miR-101-3p. In conclusion, Tp infection

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up-regulates miR-101-3p expression, which in turn inhibits the TLR2 signaling pathway,

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leading to reduced cytokines production.

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Keywords: Treponema pallidum, miR-101-3p, macrophages, TLR2, inflammatory cytokines

41 42

INTRODUCTION

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Syphilis is one of the most common sexually transmitted diseases globally and is caused by 2

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the spirochet Treponema pallidum subsp. pallidum (Tp) (Radolf et al., 2006, Lafond and

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Lukehart, 2006). Syphilis affects nearly 6 million people globally and represents a major

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public health issue (Korenromp et al., 2017). The natural history of syphilis has four stages:

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primary syphilis, secondary syphilis, latent syphilis, and tertiary syphilis (Ho and Lukehart,

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2011). Although treatment with appropriate antibiotics can improve syphilis symptoms, the

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evaluation of therapeutic response largely relies on serological tests. Patients with a greater

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than four-fold reduction in nontreponemal titers are considered as showing a good serological

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response, whereas those with less than four-fold changes in nontreponemal titers, currently

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accounting for 30–40%, are referred to as “serofast” (Seña et al., 2011). Great variation in

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symptoms and severity can occur owing to differences in host immune responses to Tp.

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Serological tests cannot distinguish syphilis in the serofast state from latent syphilis because

55

of the similar manifestations of these two subtypes.

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In recent decades, the importance of innate and adaptive immune responses in the

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immunopathogenesis of syphilis has been well appreciated. Spirochetal lipoproteins and/or

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synthetic lipopeptides can activate macrophages and dendritic cells (DCs) via CD14 and

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Toll-like receptor 1 (TLR1)/TLR2-dependent signaling pathways (Norgard et al., 1996,

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Sellati et al., 1998). Although Tp lacks lipopolysaccharide (LPS), a pro-inflammatory

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constituent of the outer membranes of Gram-negative bacteria (Ulevitch and Tobias, 1999),

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abundant lipoproteins are expressed in Tp (Chamberlain et al., 1989, Fraser et al., 1998). Both

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spirochetal lipoproteins and their synthetic analogs (lipopeptides) are potent activators of

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innate immune cells. These pathogen-associated molecular patterns (PAMPs) can bind to

65

pattern

recognition

receptors

CD14,

TLR1, 3

and

TLR2

on

the

surfaces

of

66

monocytes/macrophages and DCs, leading to activation of immune cells (Aliprantis et al.,

67

1999). Microarray analysis has shown that both TLR1 and TLR2, but not TLR6, were

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up-regulated in the skin of patients with secondary syphilis (Cruz et al., 2012), suggesting a

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crucial role of lipoprotein-TRL1 and TLR2 signaling in immune response to syphilis

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infection.

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MicroRNAs (miRNA) are short non-coding RNAs that can suppress mRNA expression

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by targeting the 3

untranslated regions (3

UTRs) of the respective mRNAs. An addition

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to their role in regulating infectious immunity, miRNAs could serve as novel molecular

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markers. Although several miRNAs, including miR-155, miR-146, and miR-21, are known to

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inhibit TLR-mediated responses upon binding of the TLR ligand to its receptor (Sheedy et al.,

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2010, Quinn and O'Neill, 2011), whether and how miRNAs regulate immunity in syphilis

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infection remains unknown. In the present study, we aimed to explore the signaling pathway

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of the immune response to Tp infection, using microarrays to obtain miRNA expression

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profiles of peripheral blood mononuclear cells (PBMCs) from syphilis patients. By

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quantitative real-time polymerase chain reaction (RT-qPCR), we verified nine differential

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miRNAs in PBMCs from syphilis patients at different stages of the disease. These miRNAs

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included miR-101-3p, miR-195-5p, and miR-223-5p, which have been shown to be

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associated with inflammation. The data also show that miR-101-3p is differentially expressed

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between syphilitic patients and healthy individuals. We verified that miR-101-3p was

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differentially expressed in CD14+ cells and used a cellular model to show how miR-101-3p

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affects expression of macrophage inflammatory cytokines. Interestingly, miR-101-3p

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expression of PBMCs was negatively correlated with plasma interleukin 6 (IL-6) levels in 4

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one individual.

89 90

RESULTS

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Expression of miR-101-3p is associated with syphilis in monocytes

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The microarray results showed that miR-101-3p may be associated with syphilis, as described

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previously (Figure S1). We first verified the differential expression of miR-101-3p. As shown

94

in Figure 1, miR-101-3p expression in PBMCs was significantly higher in patients with

95

syphilis than in healthy controls. Moreover, PBMCs from patients with serofast and primary

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syphilis exhibited expression of miR-101-3p. Interestingly, miR-101-3p expression was

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significantly higher in the serofast state than in latent syphilis. It is not clear why serofast

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state occurs after syphilitic patients treatment. Interestingly, the expression of miR-101-3p in

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the serofast state was significantly higher than that in untreated syphilitic patients and

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serologically cured (Figure 1b), suggesting the involvement of miR-101-3p in the

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development of the serofast state. In order to determine in which cells miR-101-3p was

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differentially expressed, we tested expression of miR-101-3p in the main PBMCs (Figure 1d–

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g), showing that it had similar differential expression to that of PBMCs only in CD14+ cells.

104 105

Gene ontology functional enrichment analysis of miR-101-3p target genes

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To further elucidate the role of miR-101-3p in the pathogenesis of syphilis, potential target

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genes of miR-101-3p were predicted using bioinformatics algorithms (miRWalk,

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DIANA-microT4, miRanda, miRDB, PICTAR2, and TargetScan) with miRWalk 2.0. Of the

109

42 pathways with the smallest p-values, miR-101-3p was most likely to be associated with 5

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Toll-like receptor pathways, which are known to be involved in immune regulation of

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pathogen infection (Figure 2). Studies have shown that the TLR2 and TLR4 pathways have

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important roles in bacterial infection. However, as Tp lacks the LPS ligand of TLR4, we first

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focused on how miR-101-3p regulates the TLR2 pathway. Previous studies have shown that

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TLR2 plays an important role in Tp infection. And TLR2 is one of the target genes of

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miR-101-3p. Further analysis suggested that miR-101-3p might regulate TLR2, which is

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crucial in pathogen recognition and activation of innate immunity.

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miR-101-3p down-regulates TLR2 expression during Tp infection

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As shown above, expression of miR-101-3p in CD14+ cells varied with stage of syphilis. We

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next assessed whether expression levels of TLR2 in CD14+ cells (Figure 3a) also varied with

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stage of syphilis. Indeed, expression levels of TLR2 were higher in primary and secondary

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syphilis than in either healthy controls or patients in the serofast stage (Figure 3a).

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Interestingly, TLR2 expression was significantly higher in serologically cure than serofast

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state. And the expression of miR-101-3p is opposite. To further investigate the relationships

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between miR-101-3p and TLR2, expression levels of miR-101-3p were assessed in THP-1

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cells treated with Tp. As shown in Figure 3b, miR-101-3p expression was significantly

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increased 24 h and 48 h after incubation with Tp. Expression of TLR2 was also increased at

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24 h, followed by a reduction at 48 h (Figure 3b).

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To determine the role of miR-101-3p in TLR2 expression, expression levels of TLR2

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were measured in PBMCs and THP-1 cells treated Tp, followed by incubation with either

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miR-101-3p mimic or inhibitor. As expected, the miR-101-3p mimic down-regulated 6

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expression of TLR2 mRNA and protein (Figure 3c) 48 h after incubation. Conversely, the

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miR-101-3p inhibitor up-regulated expression levels of both TLR2 mRNA and protein

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(Figure 3c). To assess whether miR-101-3p directly regulated the 3

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generated luciferase reporters encoding the normal and a mutated TLR2 3

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in Figure 3d, luciferase activity was significantly suppressed by miR-101-3p in comparison

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with controls 48 h after transfection. By contrast, luciferase activity was up-regulated by the

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miR-101-3p inhibitor. These results demonstrate that miR-101 negatively regulates TLR2

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expression.

UTR of TLR2, we UTR. As shown

140 141

miR-101-3p down-regulates the expression of inflammatory cytokines in macrophages

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Yang TC (Lin LR et al., 2018) showed that Tp promotes macrophage polarization and

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activates inflammasome pathway. As miR-101-3p down-regulates TLR2 expression of

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macrophages, we next explored whether miR-101-3p suppressed expression of inflammatory

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cytokines associated with the TLR2 signaling pathway in macrophages. In these experiments,

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we processed the cells as in Figure 4a. As expected, treatment of macrophages with Tp

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markedly increased expression levels of several pro-inflammatory cytokines, including IL-1β,

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IL-6, IL-12, and tumor necrosis factor alpha (TNF-α) (Figure 4b,c). However, Tp-induced

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up-regulation of cytokines expression was significantly inhibited by miR-101-3p mimic. By

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contrast, miR-101-3p inhibitor augmented Tp-induced up-regulation of cytokines expression.

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To determine the involvement of TLR2 in cytokines expression mediated by miR-101-3p,

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TLR small interfering RNA (TLR-siRNA) and pcDNA-3.1(-)-TLR2 were used to

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down-regulate and up-regulate TLR2, respectively. As shown in Figure 4d, silencing of TLR2 7

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greatly attenuated Tp-induced elevations in IL-1β and IL-6 mRNA levels. Conversely,

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overexpression of TLR2 increased expression levels of IL-1β and IL-6 mRNA (Figure 4f).

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We also used peptidoglycan (PGN), TLR2 agonist, instead of Tp to study the effect of

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miR-101-3p on activation of TLR2 pathway (Figure 4e). The data show that PGN can

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up-regulate the expression of IL1β, IL6 and TNF-a, but no miR-101-3p (Supplementary

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material S2). And PGN-induced up-regulation of cytokines expression was significantly

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inhibited by miR-101-3p mimics. By contrast, miR-101-3p inhibitors augmented

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PGN-induced up-regulation of cytokines expression.

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Expression of inflammatory cytokines in syphilitic patients at different stages

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As noted above, expression levels of miR-101-3p varied with the stage of syphilis and

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miR-101-3p inhibited Tp-induced production of cytokines in macrophages. Hence, we

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assessed whether circulating levels of inflammatory cytokines also varied with the stage of

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syphilis. A panel of inflammatory cytokines, including IL-1β, IL-6, IL-12, and TNF-α, in

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plasma were measured using an ELISA assay. Plasma levels of inflammatory cytokines,

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particularly IL-6 and IL-12, varied greatly with the stage of syphilis. Notably, secondary

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syphilis exhibited the highest levels of IL-6, while primary and serologically cured syphilis

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displayed the lowest levels. By contrast, expression levels of both TNF- α and IL-1β were

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similar at the different stages of syphilis.

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DISCUSSION

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After Tp infection, mammalian hosts mount robust humoral and cellular immune responses 8

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aimed at spirochetal clearance (Arroll et al., 1999, Leader et al., 2007, Leader et al., 2003,

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Salazar et al., 2007). However, Tp can escape the host immune response, resulting in

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persistent infection. So far, the mechanisms of immune regulation in Tp infection have not

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been clear. Recent studies have indicated that miRNAs participate in the pathogenesis of a

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variety of infectious diseases (Benz et al., 2016, Fu et al., 2011, Qi et al., 2012, Wang et al.,

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2015), but whether miRNAs regulate syphilis immunity remained unknown. Our previous

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study demonstrated that miR

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overexpressed in the PBMCs of syphilitic patients in comparison with healthy controls. These

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three miRNAs have the potential to regulate inflammation. A target of miR-223 is TLR4, of

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which LPS is a ligand. Considering the lack of LPS antigen in Tp, however, we did not

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initially focus on miR-233. We had also performed some experiments to study the effects of

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miR-195 on macrophage inflammation, but the results were not in line with expectations.

195

5p, miR

223

5p, and miR

101

1p were

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We demonstrated here that levels of miR-101-3p in PBMCs of syphilitic patients vary

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with subtype of syphilis, suggesting a pathogenic role of miR-101-3p in the development of

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syphilis. As PBMCs are a mixed group of various cell types, we tested expression of

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miR-101-3p in the main PBMC components to determine which of them showed differential

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expression of miR-101-3p. Interestingly, we found that miR-101-3p expression was similar to

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that of PBMCs only in CD14+ cells.

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Previous studies have demonstrated the involvement of miR-101-3p in various diseases.

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For example, genomic loss of miR-101 leads to overexpression of histone methyltransferase

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EZH2 in cancer (Varambally et al., 2008). Moreover, down-regulation of miR-101-3p is

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associated with rheumatic heart disease, an autoimmune disease provoked by S. pyogenes 9

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infection (Hai et al., 2015). The present study showed that miR-101-3p inhibited the TLR2

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pathway via targeting the TLR2 3

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function as major pro-inflammatory agonists during spirochetal infection (Salazar et al.,

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2009). TLR2 plays an important part in the inflammatory response in syphilis (Lukehart et al.,

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1980). Tp contains abundant lipoproteins, which can activate macrophages and DCs via

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CD14 and TLR1- and TLR2-dependent signaling pathways (Lien et al., 1999, Radolf et al.,

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1995). Accordingly, transcripts for both TLR1 and TLR2, but not TLR6, are up-regulated in

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the skin of patients with secondary syphilis (Cruz et al., 2012). Importantly, we demonstrated

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here that TLR2 was highly expressed in PBMCs of syphilitic patients and those who were

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serologically cured, in comparison with both healthy controls and patients in the serofast state,

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suggesting a regulatory role for TLR2 in the immune response to syphilis.

UTR in syphilis. TLRs are PAMPs and are considered to

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The TLR2 signaling pathway regulates the expression of inflammatory cytokines (Kigerl

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et al., 2010, Snodgrass et al., 2013), while TpN47 activates the TLR2 pathway and induces

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the release of pro-inflammatory cytokines, including IL-1β, IL-6, IL-12, and TNF-α

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(Brightbill et al., 1999). These cytokines can activate Th1 cells, resulting in elimination of Tp.

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IL-6 appears to be the major cytokines in syphilis immunity, with higher levels of IL-6

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observed in secondary syphilis compared with other subtypes of syphilis. Plasma levels of

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other cytokines did not differ among the subtypes of syphilis. In the present study, we also

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found a link between miR-101-3p and TRL2 in response to Tp infection. Based on these

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results, we postulate that miR-101-3p inhibits TRL2 expression, leading to a reduction in

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cytokines production (Figure 6), and that miR-101-3p levels in syphilitic patients may

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determine the levels of cytokines that result in the development of various subtypes of 10

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syphilis. For instance, in syphilitic patients, high levels of miR-101-3p decrease TLR2

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expression, resulting in a reduction in cytokines production and leading to the development of

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the serofast state. Conversely, low levels of miR-101-3p increase TLR2 expression, resulting

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in increased cytokines production and leading to a serological cure. Again, this concept

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requires further validation.

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In conclusion, miR-101 targets the TLR2 3

UTR, resulting in inhibition of TLR2

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expression, with a consequent reduction in cytokines production in Tp infection. miR-101

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negatively regulates cytokines levels and, as cytokines contribute to the elimination of Tp, it

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is likely that miR-101 levels determine the development of subtypes of syphilis.

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Measurement of miR-101 levels could facilitate differentiation between the serofast state and

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latent syphilis.

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MATERIALS AND METHODS

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Subjects

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A total of 15 healthy controls and 108 syphilitic patients, comprising 56 untreated patients

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with acute syphilis (15 primary, 18 secondary, 23 latent) and 52 patients who received

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standard treatment (28 in the serofast stage and 24 serologically cured), were enrolled in this

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study (details in Table 1).

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Collection of samples

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Peripheral blood samples were collected from patients at the STD clinic of the Dermatology

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Hospital of Southern Medical University. Diagnosis of syphilis was made according to

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Chinese syphilis prevention and treatment guidelines (version 2015). Briefly, primary syphilis 11

241

was considered to manifest as localized ulcers, whereas secondary syphilis was characterized

242

by generalized skin rashes. Latent syphilis was diagnosed by persistent positive results for a

243

specific treponemal antibody test without symptoms. Serological cure was defined as a

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positive result for a treponemal test using the Tp particle agglutination assay and nonreactive

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with a nontreponemal test such as the rapid plasma reagin test. The serofast state refers to a

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situation in which nontreponemal antibodies decline (often adequately) after treatment but

247

fail to completely revert to nonreactive. Persons with low pretreatment titers are also

248

sometimes said to be serofast when there is minimal ( 4-fold) or no change following

249

treatment. Blood samples from patients with primary, secondary, and latent syphilis were

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collected prior to treatment.

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Exclusion criteria included: (1) co-infection with human immunodeficiency virus,

252

condyloma acuminata, or other sexually transmitted diseases; (2) patients with autoimmune

253

diseases or those receiving anti-pro-inflammatory or immunosuppressive therapy, or those

254

treated with antibiotics within the past 6 months.

255

This study was approved by the Ethics Committee of the Dermatology Hospital of Southern

256

Medical University. The objectives, procedures, and potential risks were verbally explained

257

to all participants. Written informed consent was obtained from all participants prior to

258

inclusion in this study.

259

Cell culture

260

The THP1 cell line was maintained in RPMI 1640 medium (HyClone, Logan, UT, USA)

261

supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/mL

262

penicillin, and 100 µg/mL streptomycin. All cells were maintained at 37 °C and 5% CO2. For 12

263

analysis of THP-1 macrophage response to microbial ligands, log-phase cells were seeded at

264

5×105 cells/mL in a 24-well plate. Cells were stimulated with PMA (0.1 µg/mL)

265

(Sigma-Aldrich) for 24 h. Tp was obtained from vaccinated rabbit testicles. To study the role

266

of miR-101-3p in macrophages, a specific mimic and inhibitor were transfected into

267

macrophages after 24 h incubation with Tp. The supernatants of cell cultures were collected

268

for cytokines measurements.

269

Quantitative RT-PCR for analysis of miRNA

270

PBMCs were isolated from venous blood by a standard procedure of Ficoll gradient

271

centrifugation, performed according to the manufacturer's instructions. Total RNA was

272

extracted using TRIzol reagent (Invitrogen Waltham, MA, USA). RNA was quantified using

273

a NanoDrop One spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and

274

stored in RNase free water at -80 °C.

275

RT-qPCR was performed using a Mir-X miRNA RT-qPCR SYBR Kit (Takara

276

Biomedicals, Shiga, Japan) according to the manufacturer’s instructions. Briefly, each 1 ug of

277

total RNA was reversely transcribed to cDNA using the Mir-X miRNA First-Strand Synthesis

278

Kit (Takara Biomedicals, Shiga, Japan). The miRNA expression was analyzed in duplicate

279

and normalized to U6 using a LightCycler 480 (Roche Applied Science, Basel, Switzerland).

280

Fold changes in miRNA were calculated using the comparative Ct method.

281

Luciferase assays

282

The 3

283

psiCHECK-2 vector (Promega, Madison, WI). THP-1 cells were transfected with miR-101

284

mimic/inhibitor or controls, followed by co-transfection with 3

UTR of TLR2, its antisense sequence, and a mutant 3

13

UTR were cloned into the

UTR-luc or mutant 3

285

UTR-lucl for the luciferase activity assay. After 48 h incubation, cells were lysed and the

286

luciferase assay was performed using a dual luciferase assay system (Promega, Madison, WI).

287

Each experiment was performed in triplicate. The firefly luciferase activity and the Renilla

288

luciferase activity were measured by the luminometer (Promega, Madison, WI). The relative

289

luciferase activity is presented by the ratio of the firefly luciferase activity and the Renilla

290

luciferase activity (internal reference).

291

The miRNA mimics were small, chemically modified double-stranded RNAs that mimic

292

endogenous miRNAs and enable miRNA functional analysis. The miRNA inhibitors were

293

small, chemically modified single-stranded RNA molecules designed to specifically bind to

294

and inhibit endogenous miRNA molecules and enable miRNA functional analysis by

295

down-regulation of miRNA activity.

296

Overexpression and suppression of TLR2

297

An siRNA targeting TLR2 (siRNA-TLR2) with the sense sequence was synthesized by

298

RiboBio (Guangzhou, China). A scrambled siRNA from RiboBio was used as a negative

299

control. For siRNA-mediated inhibition of TLR2 gene expression, THP-1 cells were

300

transfected with TLR2 siRNA or scrambled siRNA at a final concentration of 50 nM, using

301

the RNA MAX siRNA Transfection Reagent (Invitrogen) according to the manufacturer’s

302

instructions. Silencing efficiency was estimated at protein levels by western blotting.

303

The TLR2 sequence (NCBI accession no. NM_001318787) was synthesized by the

304

Beijing Genomics Institute Company (China) and cloned into the pcDNA3.1(-) vectors. The

305

recombinant plasmid pcDNA3.1(-)-TLR2 harboring the correct TLR2 coding sequence and

306

the control vector pcDNA3.1(-) were transfected into THP-1 cells with lipofectamine LTX 14

307

(Invitrogen) for 48 h.

308

Western blotting

309

Whole-cell protein extracts were boiled in sodium dodecyl sulfate (SDS)/β-mercaptoethanol

310

sample buffer, separated by SDS-polyacrylamide gel electrophoresis, and then transferred to

311

PVDF membranes (Amersham Pharmacia Biotech, St. Albans, Herts, UK) by electrophoretic

312

transfer. Specific proteins were detected by the respective antibodies. Rabbit anti-TLR2

313

monoclonal antibody was purchased from Abcam (Cambridge, MA, USA), and the mouse

314

anti-GAPDH monoclonal antibody was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA,

315

USA).

316

peroxidase-conjugated rabbit anti-mouse or goat anti-rabbit IgG (Cell Signaling Technology,

317

Danvers, MA, USA), and GAPDH was used as a loading control.

318

ELISA

319

Inflammatory cytokine levels were determined in plasma or the supernatant of THP-1 by

320

ELISA according to the manufacturer’s instructions with modifications. IL12, TNF-α, IL-6

321

and

322

IL-1β ELISA kits were purchased from ELabscience, UK.

323

Flow Cytometry

324

PBMCs were isolated using density centrifugation from peripheral blood with Ficoll-Paque

325

PLUS (GE Healthcare), and were washed twice in phosphate buffered saline (PBS). Then,

326

these single cells were labelled with 6 antibodies in 100 µl staining buffer (1% fetal bovine

327

serum and 0.5M EDTA in PBS), and incubated in the dark at 4°C for 30 minutes. The

328

antibodies were used as following: mouse anti-human CD3 FITC, mouse anti-human CD4

The

specific

protein-antibody

complex

15

was

detected

by

horseradish

329

BV510, mouse anti-human CD8 APC, mouse anti-human CD14 BV421, mouse anti-human

330

CD11b PE and mouse anti-human CD19 APC-H7. Stained cells were washed twice in 1ml

331

PBS, and centrifuged at 400 x g for 5 minutes. After washes, these cells were resuspended in

332

PBS for sorting analysis with FACSAriaIII (BD Biosciences). The sorted CD4+ T cells,

333

CD8+ T cells, B cells and monocytes from all samples were finally used for miR-101-3p

334

expression profiles by real-time quantitative polymerase chain reaction (RT-qPCR).

335

Statistics

336

Statistical analyses were performed with the GraphPad Prism software 5.01. Data are

337

presented as mean ± standard deviation unless otherwise stated. Student’s t-test was used for

338

comparison between two groups. The correlation analysis was used for IL6 in plasma and

339

miR-101-3p expression in PBMCs. Significances among multiple groups were determined by

340

one-way variance with Tukey’s multiple comparison. A p-value less than 0.05 was considered

341

to indicate a significant difference.

342

DATA AVAILABILITY STATEMENT

343

Data that support the findings of this study are available from the corresponding author upon

344

request.

345

CONFLICT OF INTEREST

346

The authors state no conflict of interest.

347

ETHICAL APPROVAL

348

Ethical approval was obtained from the ethics committee of the Dermatology Hospital of 16

349

Southern Medical University (Chinese NO.GDDHLS-20170614).

350

CRediT Statement

351

Conceptualization: Heping Zheng and Bin Yang; Data curation: Tao Huang, Jieyi Yang and

352

Jun Zhang; Resources: Wujian Ke, Fei Zou, Chengsong Wan, LiuYuan Wang, Xiaohui Zhang,

353

Fangwen Liang and Shuqing Mei; Writing: Tao Huang; Writing - Review and Editing: Tao

354

Huang Zhili Rong and Qiwei Zhang; Funding Acquisition: Heping Zheng, Bin Yang, Tao

355

Huang and Jun Zhang; Supervision: Heping Zheng and Bin Yang; Formal Analysis and

356

Methodology: Tao Huang and Heping Zheng; Project Administration: Heping Zheng and Bin

357

Yang.

358

ACKNOWLEDGMENTS

359

Heping Zheng, Bin Yang, Fei Zou, Chengsong Wan, Zhili Rong, and Qiwei Zhang

360

contributed to the conception of the study; Jun Zhang contributed significantly to analysis

361

and manuscript preparation; Wujian Ke, Xiaohui Zhang, and Liuyuan Wang performed the

362

data analyses and wrote the manuscript; Fangwen Liang and Shuqing Mei helped perform the

363

analysis and took part in constructive discussions; and Tao Huang and Jieyi Yang carried out

364

all the experiments.

365

This work was supported by the following grants:

366

1. Guangdong Science and Technology Department 2017A020212008.

367

2. The National Natural Science Foundation of China (NSFC) 81772240.

368

3. Medical Science and Technology Foundation of Guangdong Province C2017043.

17

369

4. Medical Science and Technology Foundation of Guangdong Province A2016269.

370

5. Natural Science Foundation of Guangdong Province 2015A030310070.

371 372 373

18

374

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469

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470

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471 472

23

473

FIGURE LEGENDS

474 475

Figure 1. Expression of miR-101-3p is associated with syphilis in monocytes. (a,b)

476

Expression levels of miR-101-3p in PBMCs were assessed by RT-qPCR. a) All stages of

477

specimens. b) Syphilitic patients mean all untreated patients of primary syphilis, secondary

478

syphilis, and latent syphilis. These specimens were collected before treatment. Samples from

479

serofast and serologically cured patients were collected after treatment. (c–f) Expression

480

levels of miR-101-3p in sorted CD14+, CD4+, CD8+, and CD19+ cells. Normal controls

481

(n=8), primary syphilis (n=7), secondary syphilis (n=8), latent syphilis (n=8), serofast state

482

(n=9), and serological cure (n=10).

483 484

Figure 2. Gene ontology functional enrichment analysis of miR-101-3p target genes.

485

DIANA-miRPath v3.0 (htT. pallidum://www.microrna.gr/miRPathv3), an online software

486

suite dedicated to the assessment of miRNA regulatory roles and the identification of

487

controlled pathways, was used for this analysis. The x-axis shows the name of the pathway,

488

the y-axis indicates the count of genes, and colors represent the -log10 of the p-value.

489 490

Figure 3. miR-101-3p down-regulates expression of TLR2 during Tp infection. (a) TLR2

491

was detected in CD14+ cells by flow cytometry. Staining of PE-CD14 and APC-TLR2 in

492

whole blood after lysis of red blood cells. On the left is a representative of each group; on the

493

right are statistics for the average fluorescence intensity (mean). Normal controls (n=9),

494

primary syphilis (n=4), secondary syphilis (n=4), latent syphilis (n=4), serofast state (n=10), 24

495

and serological cure (n=8) (b) Expression levels of miR-101-3p and TLR2 in THP-1 cells

496

treated with Tp for 6, 24, and 48 h. (c) PBMCs and THP-1 cells were transfection with mimic

497

or inhibitor for 24 h, followed incubation with Tp by for 24 h. Left: mRNA levels of TLR2 in

498

PBMCs was detected by RT-qPCR. Right: mRNA and protein levels of TLR2 in THP-1 cells

499

was detected by RT-qPCR and western blot, respectively. (d) Luciferase vector with the

500

TLR2 3'UTR or a mutant sequence was transfected into THP-1 cells. Luciferase activity was

501

detected after transfection with mimic or inhibitor.

502 503

Figure 4. miR-101-3p regulates the expression of inflammatory cytokines in

504

macrophages. (a) Time points sampled for macrophages that THP-1 was induced by PMA.

505

(b) (i–v) mRNA levels of IL-1β, IL-6, TNF-α, IL-12A, and IL-12B as detected by RT-qPCR.

506

(c) (i–vi) Cell supernatants were used to detect the expression of inflammatory cytokines

507

using ELISA. (d) Above: western blot for TLR2; below: mRNA levels of IL-1β and IL-6 as

508

detected by RT-qPCR. (e) (i-iii) mRNA levels of IL-1β, IL-6 and TNF-α, as detected by

509

RT-qPCR. (vi-iv) Cell supernatants were used to detect the expression of inflammatory

510

cytokines using ELISA. (f) Above: western blot for TLR2; middle: mRNA levels of IL-1β

511

and IL-6 as detected by RT-qPCR; below: the levels of IL-1β and IL-6 of supernatant as

512

detected by Elisa.

513 514

Figure 5. Expression of inflammatory cytokines in syphilitic patients at different stages

515

(a) Expression of inflammatory factors in patients at different stages of syphilis. (b) The

516

correlation analysis was used for IL6 in plasma and miR-101-3p expression in PBMCs. And 25

517

the plasma and PBMCs come from one individual. Data are shown as mean ± SEM;

518

**P<0.01, ***P<0.001. (Control n=10, primary n=8, secondary n=13, latent n=15, serofast

519

n=10, serological cure n=8.)

520 521

Figure 6. Role of miR-101-3p in regulating cytokines expression. Tp activates the TLR2

522

signaling pathway, leading to production of proinflammatory cytokines such as IL-1β and

523

IL-6, possibly via NF-κB signaling (Manish et al., 2005, Sunhwa et al., 2009, Kigerl et al.,

524

2010). Tp can also increase the expression levels of miR-101-3p in macrophages, resulting in

525

a reduction in IL-1β and IL-6 production via inhibition of TLR2.

526 Group

Male/Female

RPR

-

1:1

1:2

TPPA

1:4

1:8

1:16

Heathy Control

8/7

15

0

0

0

0

0

0

Primary syphilis

12/3

0

3

4

4

2

1

15

Secondary

13/5

0

2

0

1

3

12

18

Latent syphilis

13/10

8

4

4

3

2

2

23

Serofast state

13/15

0

8

10

7

2

1

28

Serological cure

13/11

24

syphilis

0

Total

527

123

Table 1. Patient information collected

26

24

+