IL-10/STAT5 axis suppresses miR-140 to upregulate B7-H4 expression in RAW264.7 cells
IntroductionSchistosomiasis japonica, a zoonotic parasitic disease, induces complex immune regulation during infection. The inflammatory responses and immunosuppressive mechanisms co-exist to maintain immune homeostasis in schistosomiasis. B7-H4 is a critical immune checkpoint molecule that modulate...
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Frontiers Media S.A.
2025-08-01
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| Series: | Frontiers in Cellular and Infection Microbiology |
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| author | Dandan Zhu Guo Chen Guo Chen Pei Shen Weiliang Fan Chuxin Ji Yinong Duan Wenxi Gao Wenxi Gao |
| author_facet | Dandan Zhu Guo Chen Guo Chen Pei Shen Weiliang Fan Chuxin Ji Yinong Duan Wenxi Gao Wenxi Gao |
| author_sort | Dandan Zhu |
| collection | DOAJ |
| description | IntroductionSchistosomiasis japonica, a zoonotic parasitic disease, induces complex immune regulation during infection. The inflammatory responses and immunosuppressive mechanisms co-exist to maintain immune homeostasis in schistosomiasis. B7-H4 is a critical immune checkpoint molecule that modulates T cell activation and exerts immunosuppressive effects. Our previous investigations revealed that B7-H4 mRNA expression was elevated in mice infected with Schistosoma japonicum, with interleukin-10 (IL-10) demonstrating regulatory capacity to enhance B7-H4 expression in RAW264.7 macrophages. In this study, we further explore the mechanism underlying IL-10-mediated B7-H4 upregulation.MethodsWestern blot was performed to detect B7-H4 expression levels, both in mice infected with Schistosoma japonicum and in RAW264.7 cells stimulated with IL-10. RT-qPCR was performed to screen microRNAs (miR-140 et al.) in RAW264.7 cells stimulated with IL-10. Then dual-luciferase reporter assay was performed to confirm that miR-140 can directly bind to the 3’UTR of B7-H4. miR-140 promoter activity in RAW264.7 cells was also detected via dual-luciferase reporter assays. In addition, ChIP was performed to confirm the binding of transcription factors and miR-140 promoter.ResultsNotably, miR-140 was decreased in IL-10-treated microphages, accompanied by B7-H4 expression was upregulated. miR-140 can directly bind to the 3’UTR of B7-H4 and then inhibit the expression of B7-H4 in RAW264.7 cells. Meanwhile, miR-140 mimics can also attenuate IL-10-induced B7-H4 expression in RAW264.7 cells. Then we found that IL-10 may inhibit miR-140 promoter activity in RAW264.7 cells through transcription factors that binding to the - 576/- 94 bp region of the miR-140 promoter. Results by Western blot and ChIP further indicated that IL-10 could downregulate miR-140 promoter activity in a STAT5 dependence manner. After the sequence of STAT5 binding site within the - 456/- 446 bp region of the miR-140 promoter was mutated, IL-10 failed to suppress the activity produced by mutant miR-140 promoter.DiscussionIn summary, IL-10 can inhibit miR-140 through STAT5, thereby upregulating the expression of B7-H4 in RAW264.7 cells. This study may suggest a new mechanism underlying IL-10-mediated B7-H4 upregulation in macrophages. |
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| issn | 2235-2988 |
| language | English |
| publishDate | 2025-08-01 |
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| spelling | doaj-art-001bab3ce2ec463bb9beea026c6c6f412025-08-20T03:05:53ZengFrontiers Media S.A.Frontiers in Cellular and Infection Microbiology2235-29882025-08-011510.3389/fcimb.2025.16132971613297IL-10/STAT5 axis suppresses miR-140 to upregulate B7-H4 expression in RAW264.7 cellsDandan Zhu0Guo Chen1Guo Chen2Pei Shen3Weiliang Fan4Chuxin Ji5Yinong Duan6Wenxi Gao7Wenxi Gao8Department of Pathogen Biology, School of Medicine, Nantong University, Nantong, Jiangsu, ChinaDepartment of Pathogen Biology, School of Medicine, Nantong University, Nantong, Jiangsu, ChinaDepartment of Dermatology, Huashan Hospital, Fudan University, Shanghai, ChinaDepartment of Laboratory Medicine, Affiliated Hospital of Nantong University, Nantong, ChinaDepartment of Pathogen Biology, School of Medicine, Nantong University, Nantong, Jiangsu, ChinaDepartment of Pathogen Biology, School of Medicine, Nantong University, Nantong, Jiangsu, ChinaDepartment of Pathogen Biology, School of Medicine, Nantong University, Nantong, Jiangsu, ChinaDepartment of Pathogen Biology, School of Medicine, Nantong University, Nantong, Jiangsu, ChinaLaboratory Center, School of Educational Sciences, Nantong University, Nantong, Jiangsu, ChinaIntroductionSchistosomiasis japonica, a zoonotic parasitic disease, induces complex immune regulation during infection. The inflammatory responses and immunosuppressive mechanisms co-exist to maintain immune homeostasis in schistosomiasis. B7-H4 is a critical immune checkpoint molecule that modulates T cell activation and exerts immunosuppressive effects. Our previous investigations revealed that B7-H4 mRNA expression was elevated in mice infected with Schistosoma japonicum, with interleukin-10 (IL-10) demonstrating regulatory capacity to enhance B7-H4 expression in RAW264.7 macrophages. In this study, we further explore the mechanism underlying IL-10-mediated B7-H4 upregulation.MethodsWestern blot was performed to detect B7-H4 expression levels, both in mice infected with Schistosoma japonicum and in RAW264.7 cells stimulated with IL-10. RT-qPCR was performed to screen microRNAs (miR-140 et al.) in RAW264.7 cells stimulated with IL-10. Then dual-luciferase reporter assay was performed to confirm that miR-140 can directly bind to the 3’UTR of B7-H4. miR-140 promoter activity in RAW264.7 cells was also detected via dual-luciferase reporter assays. In addition, ChIP was performed to confirm the binding of transcription factors and miR-140 promoter.ResultsNotably, miR-140 was decreased in IL-10-treated microphages, accompanied by B7-H4 expression was upregulated. miR-140 can directly bind to the 3’UTR of B7-H4 and then inhibit the expression of B7-H4 in RAW264.7 cells. Meanwhile, miR-140 mimics can also attenuate IL-10-induced B7-H4 expression in RAW264.7 cells. Then we found that IL-10 may inhibit miR-140 promoter activity in RAW264.7 cells through transcription factors that binding to the - 576/- 94 bp region of the miR-140 promoter. Results by Western blot and ChIP further indicated that IL-10 could downregulate miR-140 promoter activity in a STAT5 dependence manner. After the sequence of STAT5 binding site within the - 456/- 446 bp region of the miR-140 promoter was mutated, IL-10 failed to suppress the activity produced by mutant miR-140 promoter.DiscussionIn summary, IL-10 can inhibit miR-140 through STAT5, thereby upregulating the expression of B7-H4 in RAW264.7 cells. This study may suggest a new mechanism underlying IL-10-mediated B7-H4 upregulation in macrophages.https://www.frontiersin.org/articles/10.3389/fcimb.2025.1613297/fullmiR-140B7-H4IL-10STAT5Schistosoma japonicum |
| spellingShingle | Dandan Zhu Guo Chen Guo Chen Pei Shen Weiliang Fan Chuxin Ji Yinong Duan Wenxi Gao Wenxi Gao IL-10/STAT5 axis suppresses miR-140 to upregulate B7-H4 expression in RAW264.7 cells Frontiers in Cellular and Infection Microbiology miR-140 B7-H4 IL-10 STAT5 Schistosoma japonicum |
| title | IL-10/STAT5 axis suppresses miR-140 to upregulate B7-H4 expression in RAW264.7 cells |
| title_full | IL-10/STAT5 axis suppresses miR-140 to upregulate B7-H4 expression in RAW264.7 cells |
| title_fullStr | IL-10/STAT5 axis suppresses miR-140 to upregulate B7-H4 expression in RAW264.7 cells |
| title_full_unstemmed | IL-10/STAT5 axis suppresses miR-140 to upregulate B7-H4 expression in RAW264.7 cells |
| title_short | IL-10/STAT5 axis suppresses miR-140 to upregulate B7-H4 expression in RAW264.7 cells |
| title_sort | il 10 stat5 axis suppresses mir 140 to upregulate b7 h4 expression in raw264 7 cells |
| topic | miR-140 B7-H4 IL-10 STAT5 Schistosoma japonicum |
| url | https://www.frontiersin.org/articles/10.3389/fcimb.2025.1613297/full |
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