THP-1 cells, a human monocyte cell line, were provided by Dr. Chuncai Gu, PhD (Nanfang Hospital, Southern Medical University, Guangzhou, China). THP-1 cells were grown in culture media, containing RPMI 1640 media (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco, Australia) and 1% penicillin-streptomycin. Cells were grown in a humidified 5% CO₂ atmosphere at 37°C. The culture media was then supplemented with 100 ng/mL phorbol 12-myristate 13-acetate (Sigma-Aldrich, St. Louis, MO, USA) and incubated for 24 h to induce the differentiation of the monocytes into M0 macrophages. The obtained macrophages were then cultured in a media without penicillin-streptomycin. The cells were split into two treatment groups cultured with TP (Nichols strain, kindly provided by Yinbo Jiang, Dermatology Hospital, Southern Medical University, Guangzhou, China) at a multiplicity of infection (MOI) of 20:1 (TP:cell) or phosphate-buffered saline (PBS) as the control group. The two treatment groups were incubated for 24 h.

YTHDF1, METTL3, SOCS3, and control siRNAs (Sangon Biotech, Shanghai, China) were transfected into THP-1 cells at a final concentration of 10 nM using RNA MAX siRNA Transfection Reagent (Invitrogen) according to the manufacturer’s instructions. THP-1 cells were incubated at 37°C in a CO₂ incubator for 48 h, and the transfection efficiency was evaluated using western blotting analysis as described below. The siRNA sequences are listed in Supplementary Table S1 .

THP-1 cells were transfected with the SOCS3-expressing plasmid (Sangon Biotech, Shanghai, China) using Lipo3000 (Invitrogen).

The Ethical Approval Board of Dermatology Hospital, Southern Medical University, China approved the use of tissue wax blocks from patients with secondary syphilis (GDDHLS-20180510). For immunofluorescence analysis of the tissues, the sections were permeabilized with 0.25% Triton X-100 for 0.5 h and blocked with 5% goat serum for 1 h. Tissue sections were then incubated with primary antibodies against CD68 (Cat. No. 66231-2-Ig, Proteintech, Wuhan, Hubei, China) and YTHDF1 (Cat. No. 17479-1-AP, Proteintech) at 4°C overnight, followed by incubation with the secondary antibody, Alexa Fluor^® 488 donkey anti-rabbit IgG (H+L) (A21206, Life Technologies) or Alexa Fluor^® 594 donkey anti-mouse IgG (H+L) (A21203, Life Technologies). The coverslips were mounted onto slides using an antifade mounting medium containing DAPI. The images were captured using a fluorescence microscope.

Levels of cytokines (TNF-α and IL-β) in the supernatant of the THP-1 cells were determined using ELISA kits (DAKEWE, Beijing, China) according to the manufacturer’s instructions.

RT-qPCR was performed to determine SOCS3 mRNA levels. Total RNA was extracted from THP-1 cells using TRIzol reagent (Life Technologies), following the manufacturer’s protocol. cDNA was synthesized using a PrimeScript RT Reagent Kit (Takara, Japan). The cDNA was then used as a template for qPCR with SYBR Green Master Mix (Takara, Japan), according to the manufacturer’s protocol. After amplification, cycle threshold (Ct) and ΔΔCt values were obtained for quantification.

Total protein from each group of cells was isolated and measured using a BCA Protein Assay Kit (EpiZyme, Shanghai). Total proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membrane was blocked with Protein-Free Rapid Blocking Buffer (EpiZyme, Shanghai) for 15 min at room temperature and incubated with the following primary antibodies overnight at 4°C: anti-METTL3 (Cat. No. 15073-1-AP, Proteintech), anti-METTL14 (Cat. No. 26158-1-AP, Proteintech), anti-YTHDF1 (Cat. No. 17479-1-AP, Proteintech), anti-SOCS3 (Cat. No. 14025-1-AP, Proteintech), anti-JAK2 (17670-1-AP, Proteintech), anti-p-JAK2 (ab32101, Abcam), anti-STAT3 (Cat. no. 60199-1-Ig, Proteintech), anti-p-STAT3 (ab267373; Abcam), and anti-GAPDH (Cat. No.60004-1-Ig, Proteintech), as a loading control. After washing thrice, the PVDF membrane was incubated with the secondary antibody for 2 h at room temperature. The membranes were visualized using chemiluminescence. The signal intensities were quantified using the ImageJ software (version 1.49).

Single-cell suspensions were prepared from cultured THP-1 cells and incubated with anti-CD206 (374208) and anti-CD86 (321110) antibodies (both from Abcam) for 30 min at 4°C for cell-surface staining. Data were recorded on a BD Celesta flow cytometer and analyzed using the FlowJo software (V10, Treestar).

The m6A levels were measured using an m6A RNA Methylation Assay Kit (Colorimetric) (Abcam, No. ab185912) according to the manufacturer’s instructions.

Total whole-cell RNA from the control and TP-infected cells was extracted using the TRIzol reagent (Invitrogen) and quantified using a Qubit^® RNA Assay Kit on a Qubit^® 2.0 fluorometer (Life Technologies, CA, USA). The RNA purity was assessed using a NanoPhotometer spectrophotometer (IMPLEN, CA, USA). The cDNA library was constructed using NEBNext^® UltraTM RNA Library Prep Kit for Illumina^® (NEB, USA). Sequencing was performed using an Illumina NovaSeq 6000 platform (Novogene, Beijing, China). High-quality reads were mapped to the human reference genome (hg38) using the HISAT2 program. Differential expression analysis of the two groups was performed using the DESeq2 package in R (1.10.1). Genes with expression levels differing between groups at an adjusted P value <0.05 were considered to be significantly differentially expressed. Sequencing was performed in three independent biological replicates.

Total RNA was extracted using TRIzol reagent (Invitrogen) and fragmented using a fragmentation buffer. The fragmented mRNA (100 ng) was then used as an input control, and the remainder of the RNA was incubated with magnetic ChIP protein A/G magnetic beads to isolate methylated RNA according to the manufacturer’s instructions (Magna MeRIP m6A Assay, Millipore, 17-10499). MeRIPed RNA was analyzed using RT-qPCR.

The procedure for RIP-qPCR was adapted from a previous study (13). THP-1 cells transfected with siNC, siMETTL3, or siYTHDF1 were washed twice with PBS, collected, and resuspended in immunoprecipitation lysis buffer. 10% of the cell lysate were saved as input, and the remaining sample was incubated with IgG antibody, YTHDF1 antibody, and protein G beads (Invitrogen) overnight at 4°C. After washing three times with wash buffer, the co-precipitated RNAs were extracted using TRIzol reagent and ethanol-precipitation with glycogen. Fold enrichment was determined by RT-qPCR.

Data were expressed as the mean ± standard deviation, and statistical significance was determined using an unpaired Student t-test for the two groups and ANOVA for multiple groups with SPSS 19.0 and GraphPad Prism 5.0. Statistical significance was set at P < 0.05.

To explore whether m6A methylation is related to the inflammatory response of macrophages in syphilis infection, we first investigated m6A levels in TP-infected macrophages. Compared to the control, TP stimulation upregulated the m6A levels ( Figure 1A ). We also investigated the expression patterns of the m6A writers (METTL3 and METTL14) and readers (YTHDF1) in TP-infected macrophages. After stimulation with TP for 24 h (MOI = 20:1), the expression levels of METTL3 and YTHDF1 were significantly elevated compared with those in the control group ( Figures 1B, C and Supplementary Figure S1 ). Similarly, immunofluorescence analysis showed upregulated expression of METTL3 and YTHDF1 in CD68+ macrophages from secondary syphilitic lesions compared with those of paired healthy skin tissue samples ( Figures 1D, E ). These data suggested that m6A methylation may participate in the pathological process of macrophages in syphilis.

Given the upregulation of YTHDF1 protein and RNA in TP-infected macrophages, we next verified its role in macrophage polarization. YTHDF1 silencing mediated by siRNA transfection markedly inhibited at least 80% of the YTHDF1 protein expression ( Supplementary Figure S2 ). Flow cytometry showed that YTHDF1 knockdown also upregulated the M1 markers (CD86 and iNOS) and downregulated the expression of M2 markers (CD206 and ARG1) ( Figures 2A, B and Supplementary Figure S3 ), which demonstrated that YTHDF1 inhibited M1 polarization and induced M2 polarization. Moreover, TP infection significantly upregulated the secretion of inflammatory cytokines (TNF-α and IL-1β) by macrophages, which was further enhanced by YTHDF1 knockdown ( Figures 2C, D ).

The RNA-sequencing results clearly showed that the mRNA expression patterns between the control and TP-infected macrophages significantly differed. Analysis of mRNA expression profiles showed that 3,110 genes were significantly dysregulated (log2-fold change > 0, P ≤ 0.05) in the TP-infected cells (859 upregulated genes and 1,251 downregulated genes) compared with those of the control group ( Supplementary Figure S4 ). Kyoto Encyclopedia of Genes and Genomes analysis revealed that the upregulated genes in TP-infected macrophages were enriched in cytokine production and inflammatory signaling pathways such as the TNF signaling pathway, along with pathways associated with infection, and endocytosis ( Figure 3A ). We also investigated the top 15 upregulated genes ( Figure 3B ), which suggested that SOCS3 may be associated with the negative regulation of the inflammatory response during TP infection. Furthermore, we predicted the association of YTHDF1 and SOCS3 with binding and perturbation using the m6A2Target tool ¹ ( Figure 3C ). Given that YTHDF1 functions by binding to m6A-methylated mRNA to promote its translation (9, 14), the potential m6A sites on SOCS3 mRNA were analyzed using m6Avar, demonstrating high confidence for position 172 of SOCS3 mRNA as an m6A site ( Figure 3D ).

Indeed, the results of MeRIP-qPCR confirmed that TP infection increased m6A levels in SOCS3 mRNA ( Figure 4A ). Furthermore, RIP-qPCR analysis revealed that SOCS3 mRNA is a target of YTHDF1 protein in TP-infected THP-1 cells ( Figure 4B ). To ascertain whether SOCS3 mRNA is a substrate for YTHDF1, we further examined the influence of YTHDF1 on the translation of SOCS3. Western blot analysis demonstrated that the SOCS3 expression level was elevated in the THP-1 cells after stimulation with TP ( Figure 4C ). YTHDF1 knockdown did not affect SOCS3 mRNA expression ( Figure 4D ); however, knockdown of YTHDF1 significantly decreased the protein expression of SOCS3 ( Figures 4E, F ). Treatment with cycloleucine, an m6A methylation inhibitor, negatively regulated SOCS3 expression in the cells ( Figures 4G, H ). Furthermore, SOSC3 protein levels were elevated in THP-1 cells with YTHDF1 overexpression compared to those in control cells but were reduced after cycloleucine pretreatment ( Figure 4I ). In addition, we investigated whether METTL3 affects the m6A modification of SOCS3 mRNA. Western blotting showed that siRNA effectively repressed the expression of METTL3 ( Supplementary Figure S5 ). Further MeRIP-qPCR analysis showed that, compared with that in the control group, the m6A modification level of SOCS3 mRNA was decreased in the METTL3-knockdown group ( Figure 4J ). Besides, the m6A levels in the SOCS3 mRNA were elevated in the THP-1 cells with METTL3 overexpression compared to those in control cells but were reduced after cycloleucine pretreatment ( Figure 4K ). Silencing METTL3 impaired the protein expression of SOCS3 ( Figures 4L, M ). Protein levels of SOSC3 were elevated in the THP-1 cells with METTL3 overexpression compared to those in control cells but were reduced after cycloleucine pretreatment ( Figure 4N ). Collectively, these results indicate that METTL3 promotes the m6A modification of SOCS3 mRNA, and that YTHDF1 recognizes the m6A-modified SOCS3 mRNA to promote its translation.

Previous studies have demonstrated that SOCS3 is an important negative regulator of JAK2–STAT3 signaling, which plays an indispensable role in regulating the inflammatory response (9, 14). Based on the above findings, we investigated whether SOCS3 negatively affects the inflammatory response by inhibiting JAK2–STAT3 signaling in macrophages infected with TP. Compared with those of control cells, the secretion levels of inflammatory cytokines (TNF-α and IL-1β) increased in SOCS3-knockdown cells infected with TP ( Figures 5A, B ). Furthermore, silencing of SOCS3 promoted the activation of JAK2–STAT3 signaling ( Figures 5C, D ). The overexpression of SOCS3 partially rescued the activation of JAK2–STAT3 signaling caused by YTHDF1 knockdown ( Figure 5E ).