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YTHDF1 Negatively Regulates Treponema pallidum-Induced Inflammation in THP-1 Macrophages by Promoting SOCS3 Translation in an m6A-Dependent Manner

Li, Zhijia, Teng, Muzhou, Jiang, Yinbo, Zhang, Litian, Luo, Xi, Liao, Yuhui, Yang, Bin·2022·Frontiers in Immunology
DOI 10.3389/fimmu.2022.857727PMC PMC9013966
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Abstract

Background: Previous studies have confirmed that the bacterium Treponema pallidum (TP) or its proteins provide signals to macrophages that induce an inflammatory response; however, little is known about the negative regulation of this macrophage-mediated inflammatory response during syphilis infection or the underlying mechanism. Recent evidence suggests the role of the RNA modification, N 6-adenosine methylation (m6A), in regulating the inflammatory response and pathogen-host cell interactions. Therefore, we hypothesized that m6A plays a role in the regulation of the inflammatory response in macrophages exposed to TP. Methods: We first assessed m6A levels in TP-infected macrophages differentiated from the human monocyte cell line THP-1. The binding and interaction between the m6A “writer” methyltransferase-like 3 (METTL3) or the m6A “reader” YT521-B homology (YTH) domain-containing protein YTHDF1 and the suppressor of cytokine signaling 3 (SOCS3), as a major regulator of the inflammatory response, were explored in differentiated TP-infected THP-1 cells as well as in secondary syphilitic lesions from patients. The mechanisms by which YTHDF1 and SOCS3 regulate the inflammatory response in macrophages were assessed. Results and Conclusion: After macrophages were stimulated by TP, YTHDF1 was upregulated in the cells. YTHDF1 was also upregulated in the syphilitic lesions compared to adjacent tissue in patients. YTHDF1 recognizes and binds to the m6A methylation site of SOCS3 mRNA, consequently promoting its translation, thereby inhibiting the JAK2/STAT3 pathway, and reducing the secretion of inflammatory factors, which results in anti-inflammatory regulation. This study provides the first demonstration of the role of m6A methylation in the pathological process of syphilis and further offers new insight into the pathogenesis of TP infection.

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Figure 1 m6A methylation: the m6A writer METTL3 and the reader YTHDF1 are upregulated in TP-infected macrophages. (A) Colorimetric quantification of m6A methylation in RNA from THP-1 cells with or without TP infection. (B, C) Western blot analysis of METTL3, METTL14, and YTHDF1 in THP-1 cells with or without TP infection. GAPDH was used as a control. *P < 0.05, **P < 0.01. (D, E) Immunofluorescence analysis of YTHDF1 or METTL3 in CD68+ macrophages in secondary syphilitic lesions and paired non-lesional skin tissue. Scale bar: 20 μm or 5 μm.
Figure 1 m6A methylation: the m6A writer METTL3 and the reader YTHDF1 are upregulated in TP-infected macrophages. (A) Colorimetric quantification of m6A methylation in RNA from THP-1 cells with or without TP infection. (B, C) Western blot analysis of METTL3, METTL14, and YTHDF1 in THP-1 cells with or without TP infection. GAPDH was used as a control. *P < 0.05, **P < 0.01. (D, E) Immunofluorescence analysis of YTHDF1 or METTL3 in CD68+ macrophages in secondary syphilitic lesions and paired non-lesional skin tissue. Scale bar: 20 μm or 5 μm.CC BY · PubMed Central
Figure 2 YTHDF1 regulates M2 macrophage polarization and suppresses the secretion of inflammatory cytokines in THP-1 cells infected with TP. (A, B) Flow cytometry profile of the expression of M1 and M2 markers (CD86 and CD206) in THP-1 cells infected with TP. (C-D) The concentrations of inflammatory cytokines (TNF-α and IL-1β) in the supernatants of THP-1 cells treated with TP. Data are shown as the mean ± S.D. **P < 0.01, ***P < 0.001,****P < 0.0001.
Figure 2 YTHDF1 regulates M2 macrophage polarization and suppresses the secretion of inflammatory cytokines in THP-1 cells infected with TP. (A, B) Flow cytometry profile of the expression of M1 and M2 markers (CD86 and CD206) in THP-1 cells infected with TP. (C-D) The concentrations of inflammatory cytokines (TNF-α and IL-1β) in the supernatants of THP-1 cells treated with TP. Data are shown as the mean ± S.D. **P < 0.01, ***P < 0.001,****P < 0.0001.CC BY · PubMed Central
Figure 3 SOCS3 may be the target of the m6A reader YTHDF1 in TP-infected macrophages. (A) KEGG pathway analysis of biological processes associated with inflammation and infection in the RNA-seq dataset. (B) Heat map of the top 15 significantly upregulated mRNAs in TP-infected macrophages compared with control cells. (C) The association of binding and perturbation between YTHDF1 and SOCS3 was predicted by m6A2Target. (D) The potential m6A sites on SOCS3 mRNA were analyzed by m6Avar.
Figure 3 SOCS3 may be the target of the m6A reader YTHDF1 in TP-infected macrophages. (A) KEGG pathway analysis of biological processes associated with inflammation and infection in the RNA-seq dataset. (B) Heat map of the top 15 significantly upregulated mRNAs in TP-infected macrophages compared with control cells. (C) The association of binding and perturbation between YTHDF1 and SOCS3 was predicted by m6A2Target. (D) The potential m6A sites on SOCS3 mRNA were analyzed by m6Avar.CC BY · PubMed Central
Figure 4 YTHDF1 and METTL3 regulate SOCS3 translation in an m6A-dependent manner. (A) MeRIP-qPCR analysis of the m6A levels of SOCS3 mRNA in THP-1 cells with or without TP infection. Total RNA was isolated from the NC group or TP-infected group. Methylated RNA was immunoprecipitated and then RT-qPCR analysis of the immunoprecipitated RNA was performed. (B) RIP analysis of the interaction of SOCS3 mRNA with YTHDF1 protein in THP-1 cells with or without TP infection. Enrichment of SOCS3 with YTHDF1 was assessed by RT-PCR and normalized to the input. (C) Western blot analysis of SOCS3 in THP-1 cells with or without TP infection. GAPDH was used as a control. (D) RT-PCR analysis of SOCS3 mRNA in THP-1 cells with or without TP infection following transfection with siNC or siYTHDF1. *P < 0.05 compared with siNC group. (E, F) Western blot analysis of SOCS3 in THP-1 cells with or without TP infection following transfection with siNC or siYTHDF1. GAPDH was used as a control. (G, H) Western blot analysis of SOCS3 in THP-1 cells with or without TP infection and with or without cycloleucine treatment. (I) Western blot analysis of SOCS3 in THP-1 cells transfected with control or YTHDF1 plasmid. (J) MeRIP-qPCR analysis of the m6A levels of SOCS3 mRNA in TP infected-THP-1 cells with or without METTL3 knockdown. (K) MeRIP-qPCR analysis of the m6A levels of SOCS3 mRNA in THP-1 cells transfected with control or YTHDF1 plasmid. (L, M) Western blot analysis of SOCS3 in THP-1 cells with or without TP infection following transfection with control or METTL3 knockdown. (N) Western blot analysis of SOCS3 in THP-1 cells transfected with control or METTL3 plasmid. *P < 0.05, **P < 0.01, ***P < 0.001; ns, no significance.
Figure 4 YTHDF1 and METTL3 regulate SOCS3 translation in an m6A-dependent manner. (A) MeRIP-qPCR analysis of the m6A levels of SOCS3 mRNA in THP-1 cells with or without TP infection. Total RNA was isolated from the NC group or TP-infected group. Methylated RNA was immunoprecipitated and then RT-qPCR analysis of the immunoprecipitated RNA was performed. (B) RIP analysis of the interaction of SOCS3 mRNA with YTHDF1 protein in THP-1 cells with or without TP infection. Enrichment of SOCS3 with YTHDF1 was assessed by RT-PCR and normalized to the input. (C) Western blot analysis of SOCS3 in THP-1 cells with or without TP infection. GAPDH was used as a control. (D) RT-PCR analysis of SOCS3 mRNA in THP-1 cells with or without TP infection following transfection with siNC or siYTHDF1. *P < 0.05 compared with siNC group. (E, F) Western blot analysis of SOCS3 in THP-1 cells with or without TP infection following transfection with siNC or siYTHDF1. GAPDH was used as a control. (G, H) Western blot analysis of SOCS3 in THP-1 cells with or without TP infection and with or without cycloleucine treatment. (I) Western blot analysis of SOCS3 in THP-1 cells transfected with control or YTHDF1 plasmid. (J) MeRIP-qPCR analysis of the m6A levels of SOCS3 mRNA in TP infected-THP-1 cells with or without METTL3 knockdown. (K) MeRIP-qPCR analysis of the m6A levels of SOCS3 mRNA in THP-1 cells transfected with control or YTHDF1 plasmid. (L, M) Western blot analysis of SOCS3 in THP-1 cells with or without TP infection following transfection with control or METTL3 knockdown. (N) Western blot analysis of SOCS3 in THP-1 cells transfected with control or METTL3 plasmid. *P < 0.05, **P < 0.01, ***P < 0.001; ns, no significance.CC BY · PubMed Central
Figure 5 SOCS3 suppresses inflammation in TP-infected macrophages via the JAK2-STAT3 pathway. (A, B) The concentrations of inflammatory cytokines (TNF-α and IL-1β) in the supernatants of THP-1 cells with or without TP infection following transfection with siNC or siSOCS3. (C, D) Western blot analysis of SOCS3, p-JAK2, JAK2, p-STAT3, and STAT3 in THP-1 cells transfected with siNC or siSOCS3. (E) Western blot analysis of SOCS3, p-JAK2, JAK2, p-STAT3, and STAT3 in piPSCs with or without YTHDF1 knockdown and transfected with control or SOCS3 plasmid. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Figure 5 SOCS3 suppresses inflammation in TP-infected macrophages via the JAK2-STAT3 pathway. (A, B) The concentrations of inflammatory cytokines (TNF-α and IL-1β) in the supernatants of THP-1 cells with or without TP infection following transfection with siNC or siSOCS3. (C, D) Western blot analysis of SOCS3, p-JAK2, JAK2, p-STAT3, and STAT3 in THP-1 cells transfected with siNC or siSOCS3. (E) Western blot analysis of SOCS3, p-JAK2, JAK2, p-STAT3, and STAT3 in piPSCs with or without YTHDF1 knockdown and transfected with control or SOCS3 plasmid. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.CC BY · PubMed Central
Figure 6 A schematic diagram revealing the proposed mechanism by which YTHDF1 mediates the protein expression of SOCS3 in an m6A-dependent manner and regulates the inflammatory response in macrophages infected with TP.
Figure 6 A schematic diagram revealing the proposed mechanism by which YTHDF1 mediates the protein expression of SOCS3 in an m6A-dependent manner and regulates the inflammatory response in macrophages infected with TP.CC BY · PubMed Central

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Citations

Cites

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Metadata

Authors
Li, Z., Teng, M., Jiang, Y., Zhang, L., Luo, X., Liao, Y., & Yang, B.
Venue
Frontiers in Immunology
Publisher
Frontiers Media SA
Published
2022-04-04
Volume
13
ISSN
1664-3224
DOI
10.3389/fimmu.2022.857727
PMC
PMC9013966
Cited by
39
References
2
Licence
creativecommons.org/licenses/by/4.0/ (vor)
Funders
National Natural Science Foundation of China (81772240)

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