MiR‐342 controls Mycobacterium tuberculosis susceptibility by modulating inflammation and cell death
2021; Springer Nature; Volume: 22; Issue: 9 Linguagem: Inglês
10.15252/embr.202052252
ISSN1469-3178
AutoresBeibei Fu, Xiaoyuan Lin, Shun Tan, Rui Zhang, Weiwei Xue, Haiwei Zhang, Shanfu Zhang, Qingting Zhao, Yu Wang, Kelly Feldman, Lei Shi, Shaolin Zhang, Weiqi Nian, Krishna Chaitanya Pavani, Zhifeng Li, Xingsheng Wang, Haibo Wu,
Tópico(s)Mycobacterium research and diagnosis
ResumoArticle20 July 2021free access Source DataTransparent process MiR-342 controls Mycobacterium tuberculosis susceptibility by modulating inflammation and cell death Beibei Fu Beibei Fu School of Life Sciences, Chongqing University, Chongqing, ChinaThese authors contributed equally to this work. Search for more papers by this author Xiaoyuan Lin Xiaoyuan Lin School of Life Sciences, Chongqing University, Chongqing, ChinaThese authors contributed equally to this work. Search for more papers by this author Shun Tan Shun Tan Chongqing Public Health Medical Center, Chongqing, ChinaThese authors contributed equally to this work. Search for more papers by this author Rui Zhang Rui Zhang Department of Respiratory Medicine, First Affiliated Hospital of Chongqing Medical University, Chongqing, ChinaThese authors contributed equally to this work. Search for more papers by this author Weiwei Xue Weiwei Xue School of Pharmaceutical Sciences, Chongqing University, Chongqing, China Search for more papers by this author Haiwei Zhang Haiwei Zhang Chongqing Key Laboratory of Translational Research for Cancer Metastasis and Individualized Treatment, Chongqing University Cancer Hospital, Chongqing, China Search for more papers by this author Shanfu Zhang Shanfu Zhang School of Life Sciences, Chongqing University, Chongqing, China Search for more papers by this author Qingting Zhao Qingting Zhao School of Life Sciences, Chongqing University, Chongqing, China Search for more papers by this author Yu Wang Yu Wang Technical Center of Chongqing Customs, Chongqing, China Search for more papers by this author Kelly Feldman Kelly Feldman Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA Search for more papers by this author Lei Shi Lei Shi School of Life Sciences, Chongqing University, Chongqing, China Search for more papers by this author Shaolin Zhang Shaolin Zhang School of Pharmaceutical Sciences, Chongqing University, Chongqing, China Search for more papers by this author Weiqi Nian Weiqi Nian Chongqing Key Laboratory of Translational Research for Cancer Metastasis and Individualized Treatment, Chongqing University Cancer Hospital, Chongqing, China Search for more papers by this author Krishna Chaitanya Pavani Krishna Chaitanya Pavani Department of Nutrition, Genetics and Ethology, Ghent University, Merelbeke, Belgium Search for more papers by this author Zhifeng Li Corresponding Author Zhifeng Li [email protected] orcid.org/0000-0001-5668-7567 School of Life Sciences, Chongqing University, Chongqing, China Chongqing Center for Disease Control and Prevention, Chongqing, China Search for more papers by this author Xingsheng Wang Corresponding Author Xingsheng Wang [email protected] orcid.org/0000-0002-8190-7506 Department of Respiratory Medicine, Chongqing Emergency Medical Center, Affiliated Central Hospital of Chongqing University, Chongqing, China Search for more papers by this author Haibo Wu Corresponding Author Haibo Wu [email protected] orcid.org/0000-0002-5961-2947 School of Life Sciences, Chongqing University, Chongqing, China Search for more papers by this author Beibei Fu Beibei Fu School of Life Sciences, Chongqing University, Chongqing, ChinaThese authors contributed equally to this work. Search for more papers by this author Xiaoyuan Lin Xiaoyuan Lin School of Life Sciences, Chongqing University, Chongqing, ChinaThese authors contributed equally to this work. Search for more papers by this author Shun Tan Shun Tan Chongqing Public Health Medical Center, Chongqing, ChinaThese authors contributed equally to this work. Search for more papers by this author Rui Zhang Rui Zhang Department of Respiratory Medicine, First Affiliated Hospital of Chongqing Medical University, Chongqing, ChinaThese authors contributed equally to this work. Search for more papers by this author Weiwei Xue Weiwei Xue School of Pharmaceutical Sciences, Chongqing University, Chongqing, China Search for more papers by this author Haiwei Zhang Haiwei Zhang Chongqing Key Laboratory of Translational Research for Cancer Metastasis and Individualized Treatment, Chongqing University Cancer Hospital, Chongqing, China Search for more papers by this author Shanfu Zhang Shanfu Zhang School of Life Sciences, Chongqing University, Chongqing, China Search for more papers by this author Qingting Zhao Qingting Zhao School of Life Sciences, Chongqing University, Chongqing, China Search for more papers by this author Yu Wang Yu Wang Technical Center of Chongqing Customs, Chongqing, China Search for more papers by this author Kelly Feldman Kelly Feldman Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA Search for more papers by this author Lei Shi Lei Shi School of Life Sciences, Chongqing University, Chongqing, China Search for more papers by this author Shaolin Zhang Shaolin Zhang School of Pharmaceutical Sciences, Chongqing University, Chongqing, China Search for more papers by this author Weiqi Nian Weiqi Nian Chongqing Key Laboratory of Translational Research for Cancer Metastasis and Individualized Treatment, Chongqing University Cancer Hospital, Chongqing, China Search for more papers by this author Krishna Chaitanya Pavani Krishna Chaitanya Pavani Department of Nutrition, Genetics and Ethology, Ghent University, Merelbeke, Belgium Search for more papers by this author Zhifeng Li Corresponding Author Zhifeng Li [email protected] orcid.org/0000-0001-5668-7567 School of Life Sciences, Chongqing University, Chongqing, China Chongqing Center for Disease Control and Prevention, Chongqing, China Search for more papers by this author Xingsheng Wang Corresponding Author Xingsheng Wang [email protected] orcid.org/0000-0002-8190-7506 Department of Respiratory Medicine, Chongqing Emergency Medical Center, Affiliated Central Hospital of Chongqing University, Chongqing, China Search for more papers by this author Haibo Wu Corresponding Author Haibo Wu [email protected] orcid.org/0000-0002-5961-2947 School of Life Sciences, Chongqing University, Chongqing, China Search for more papers by this author Author Information Beibei Fu1, Xiaoyuan Lin1, Shun Tan2, Rui Zhang3, Weiwei Xue4, Haiwei Zhang5, Shanfu Zhang1, Qingting Zhao1, Yu Wang6, Kelly Feldman7, Lei Shi1, Shaolin Zhang4, Weiqi Nian5, Krishna Chaitanya Pavani8, Zhifeng Li *,1,9, Xingsheng Wang *,10 and Haibo Wu *,1 1School of Life Sciences, Chongqing University, Chongqing, China 2Chongqing Public Health Medical Center, Chongqing, China 3Department of Respiratory Medicine, First Affiliated Hospital of Chongqing Medical University, Chongqing, China 4School of Pharmaceutical Sciences, Chongqing University, Chongqing, China 5Chongqing Key Laboratory of Translational Research for Cancer Metastasis and Individualized Treatment, Chongqing University Cancer Hospital, Chongqing, China 6Technical Center of Chongqing Customs, Chongqing, China 7Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA 8Department of Nutrition, Genetics and Ethology, Ghent University, Merelbeke, Belgium 9Chongqing Center for Disease Control and Prevention, Chongqing, China 10Department of Respiratory Medicine, Chongqing Emergency Medical Center, Affiliated Central Hospital of Chongqing University, Chongqing, China **Corresponding author. Tel: +86 23 68812969; E-mail: [email protected] ***Corresponding author. Tel: +86 23 63692253; E-mail: [email protected] ****Corresponding author. Tel: +86 23 65678491; E-mail: [email protected] EMBO Reports (2021)22:e52252https://doi.org/10.15252/embr.202052252 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb) that places a heavy strain on public health. Host susceptibility to Mtb is modulated by macrophages, which regulate the balance between cell apoptosis and necrosis. However, the role of molecular switches that modulate apoptosis and necrosis during Mtb infection remains unclear. Here, we show that Mtb-susceptible mice and TB patients have relatively low miR-342-3p expression, while mice with miR-342-3p overexpression are more resistant to Mtb. We demonstrate that the miR-342-3p/SOCS6 axis regulates anti-Mtb immunity by increasing the production of inflammatory cytokines and chemokines. Most importantly, the miR-342-3p/SOCS6 axis participates in the switching between Mtb-induced apoptosis and necrosis through A20-mediated K48-linked ubiquitination and RIPK3 degradation. Our findings reveal several strategies by which the host innate immune system controls intracellular Mtb growth via the miRNA-mRNA network and pave the way for host-directed therapies targeting these pathways. Synopsis The miR-342/SOCS6 axis promotes anti-Mtb defense by increasing the production of inflammatory factors, and by switching cell death pathways from necrosis to apoptosis through A20 mediated K48 ubiquitination and degradation of RIPK3. The miR-342/SOCS6 axis controls susceptibility to Mycobacterium tuberculosis infection in mouse and human. The miR-342/SOCS6 axis regulates cell death in Mtb-infected macrophages. Ubiquitination-dependent degradation of RIPK3 is decisive for the Mtb-resistance phenotype. Introduction Tuberculosis (TB) is a globally distributed infectious disease caused by Mycobacterium tuberculosis (Mtb). According to recent studies, more than one-quarter of the global human population has been infected with Mtb (Chakaya et␣al, 2021). However, only about 10% of infected individuals develop active TB, indicating that innate immunity likely plays a critical role in limiting Mtb replication. MicroRNAs (miRNAs) act by negatively regulating the expression of key genes (Bartel, 2018). Some evidence indicates that host miRNAs may impact the microbial life cycle and pathogenesis (Jopling et␣al, 2005; Huang et␣al, 2007; Liu et␣al, 2016). More commonly, bacteria can regulate the expression of host-specific miRNAs and weaken the host's immunity to promote survival and immune evasion (Kumar et␣al, 2015; Liu et␣al, 2018; Fu et␣al, 2020a). Recently, it has also been reported that microbe-derived miRNAs have a negative effect on host antimicrobial immunity (Sullivan et␣al, 2005; Choy et␣al, 2008). Thus, both bacterial and host miRNAs are likely to influence the relationship between hosts and pathogens. It has been shown that miR-342-3p is involved in the immune response in a variety of diseases. For example, overexpression of miR-342-3p can greatly suppress the inflammatory response and lipid uptake in THP-1 cells and can therefore regulate the development of atherosclerosis (Wang et␣al, 2019). In a recent study, miR-342-3p was shown to play an anti-inflammatory role in regulatory T cells and to contribute to glucocorticoid-mediated treatment of inflammation in murine autoimmune models (Kim et␣al, 2020). However, the role of miR-342-3p in anti-tuberculosis immunity has not yet been reported. SOCS proteins are negative regulators of the JAK/STAT pathway (Alexander & Hilton, 2004), and accumulating evidence indicates that they are involved in the control of the cytokine networks responsible for adequate and efficient innate immune responses (Yoshimura et␣al, 2007). For example, SOCS1 expression is significantly increased in patients with tuberculosis, where SOCS1 transcript levels are correlated with disease severity (Masood et␣al, 2012; Masood et␣al, 2013; Masood et␣al, 2014). SOCS1, 4, and 5 are highly expressed in mice infected with hypervirulent Mtb (Manca et␣al, 2005). CISH, a founding member of the SOCS family, can mediate control of Mtb in mice shortly after infection (Carow et␣al, 2017). Meanwhile, SOCS6 expression is reportedly enhanced by exosomes from IL-1β-primed mesenchymal stem cells; this process is related to osteoarthritic signal regulation (Kim et␣al, 2021). In addition, the homologs of vertebrate SOCS6 in Eriocheir sinensis are critical to the immune response to bacterial and viral infection (Qu et␣al, 2018). These studies suggest that SOCS6 may also play a role in inflammation and infection. However, to our knowledge, there are no currently published studies on the role of SOCS6 in the pathogenesis of tuberculosis. C3HeB/FeJ (C3H) and C57BL/6J (B6) inbred mice are often used as models in TB susceptibility studies. C3H mice are extremely susceptible to virulent Mtb, have marked lung pathology, and die soon after infection with Mtb. In contrast, B6 mice, a substrain derived from C57BL/6 mice, have been reported to be resistant to Mtb (Kramnik et␣al, 2000; Pan et␣al, 2005; Kramnik, 2008). More specifically, after Mtb stimulation, C3H mouse bone marrow-derived macrophages (BMDMs) showed characteristic necrosis, whereas B6 BMDMs mainly underwent apoptosis. Previous studies also showed that extreme susceptibility to Mtb was associated with necrosis of Mtb-susceptible macrophages (Kramnik et␣al, 2000; Pan et␣al, 2005; Wu et␣al, 2015; Leu et␣al, 2017). The precise mechanism for this phenomenon, however, is not well understood. In this study, we used C3H and B6 inbred mice to demonstrate that miR-342-3p targets and represses Socs6, a negative regulator of cytokine signaling. We showed that the miR-342-3p/SOCS6 axis modulates anti-Mtb immunity by inducing production of inflammatory cytokines (TNF-α, IL-1, IL-6, and CXCL15) and chemokines (CCL5, CXCL10, and ICAM1). Most importantly, the miR-342-3p/SOCS6 axis is involved in the switching between Mtb-induced apoptosis and necrosis. Collectively, we have demonstrated a new regulatory pathway involved in tissue necrosis during Mtb infection. These findings suggest that new host-based therapies targeting these pathways might help combat drug-susceptible and drug-resistant TB. Results MiR-342-3p is associated with TB susceptibility Our group has previously reported that array-based miRNA profiling can help to shed light on the role of miRNAs in the switching between Mtb-induced apoptosis and necrosis (Wu et␣al, 2016). We noticed that in macrophages with an Mtb-resistant phenotype, two of the five highly expressed miRNAs, miR-342-5p, and miR-342-3p, were spliced from the same precursor (NCBI Accession No. PRJNA279232). Using C3H and B6 BMDMs, we first investigated whether miR-342 expression was correlated with Mtb infection in mouse macrophages. Semiquantitative RT–PCR analysis showed that the expression levels of primary (pri-), precursor (pre-), and mature miR-342 transcripts were upregulated in Mtb-stimulated B6 BMDMs but were not found in C3H BMDMs (Fig 1A). The relative levels of mature miR-342-5p and miR-342-3p in B6 and C3H BMDMs were then examined through northern blotting and quantitative RT–PCR (qRT–PCR). The results showed that miR-342-5p and miR-342-3p expression was much higher in B6 BMDMs than in C3H BMDMs (Fig 1B and C). Figure 1. MiR-342-3p associates with TB susceptibility A. Expression changes of pri-, pre-, and mature miR-342 transcripts were analyzed by semiquantitative PCR in C3H or B6 BMDMs after Mtb infection. Representative blots from n = 3 biological replicates are shown. B, C. The relative expressions of miR-342-3p and miR-342-5p were analyzed by northern blotting (B, representative blots from n = 3 biological replicates are shown) or quantitative real-time PCR (C, data are shown as the mean ± SEM of n = 3 biological replicates) in Mtb-stimulated C3H and B6 BMDMs. D–G. Cell death mechanisms of C3H BMDMs transfected with miR-342-3p mimic (D), or B6 BMDMs transfected with miR-342-3p inhibitor (F), followed by Mtb infection for 36 h. Cell viabilities of C3H BMDMs transfected with miR-342-3p mimic (E), or B6 BMDMs transfected with miR-342-3p inhibitor (G), followed by stimulation with Mtb or z-VAD (20 μM) for 24 h. Data are shown as the mean ± SEM of n = 3 biological replicates. H, I. Mtb growth rates of C3H BMDMs transfected with miR-342-3p mimic (H), or B6 BMDMs transfected with miR-342-3p inhibitor (I) after Mtb infection. Data are shown as the mean ± SEM of n = 3 biological replicates. J. Expression levels of miR-342-3p in PBMCs from healthy controls (n = 27) or TB patients (n = 34) were detected by qRT–PCR. Data are shown as the medians ± interquartile ranges. Data information: ANOVA followed by Bonferroni post hoc test (C-I) and Mann–Whitney U test (J) were used for data analysis. *P < 0.05, **P < 0.01. Abbreviation: NC, negative control. Source data are available online for this figure. Source Data for Figure␣1D [embr202052252-sup-0003-SDataFig1.xlsx] Download figure Download PowerPoint Next, a microRNA mimic and inhibitor were used to investigate whether miR-342 was related to the Mtb-resistant phenotype. The mechanism of cell death in Mtb-infected wild-type C3H and B6 BMDMs is verified in Fig EV1A, and the effectiveness of the miR-342-3p mimic and inhibitor is verified in Fig EV1B. Transfection of C3H BMDMs with the miR-342-3p mimic switched Mtb-induced necrosis to caspase-dependent cell apoptosis; this switching was inhibited by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD, a pan-caspase inhibitor (Zhang et␣al, 2009)) (Figs 1D and E, and EV1C). On the other hand, inhibition of miR-342-3p in B6 BMDMs turned Mtb-induced apoptosis into caspase-independent necrosis (Figs 1F and EV1D), and cells treated with both Mtb and zVAD showed decreased cell viability (Fig 1G). Furthermore, transfection of C3H BMDMs with the miR-342-3p mimic significantly hindered Mtb growth (Fig 1H), while inhibition of miR-342-3p in B6 BMDMs increased Mtb survival and replication (Fig 1I). In addition, treatment of C3H BMDMs with the miR-342-3p inhibitor resulted in more severe necrosis, while treatment of B6 BMDMs with the miR-342-3p mimic resulted in a greater degree of apoptosis (Fig EV1-EV4). Since the other splicing product, miR-342-5p, showed no significant effect on the mechanisms of cell death (Fig EV1I and J), we chose to further investigate miR-342-3p. As miR-342-3p is conserved between mice and humans, we analyzed the production of miR-342-3p in peripheral blood mononuclear cells (PBMCs) from 34 TB patients. We observed that TB patients expressed relatively low levels of miR-342-3p (Fig 1J). These results suggest that miR-342-3p may act as a positive regulator of the immune response to Mtb infection. Click here to expand this figure. Figure EV1. MiR-342-3p is associated with TB susceptibility A. Cell death mechanisms of C3H and B6 BMDMs after stimulation with Mtb for 36 h. Data are shown as the mean ± SEM of n = 3 biological replicates. B. Relative miRNA expression was detected by qRT–PCR using miR-342-3p specific primer. Data are shown as the mean ± SEM of n = 3 biological replicates. C, D. Cell death mechanisms of C3H BMDMs transfected with miR-342-3p mimic (C), or B6 BMDMs transfected with miR-342-3p inhibitor (D), followed by Mtb infection for 36 h. Representative data (from n = 3 biological replicates) are shown as the mean ± SEM of technical replicates. E, F. Cell death mechanisms of C3H BMDMs transfected with miR-342-3p inhibitor (E), or B6 BMDMs transfected with miR-342-3p mimic (F), followed by Mtb infection for 36 h. Data are shown as the mean ± SEM of n = 3 biological replicates. G, H. Mtb growth rates of C3H BMDMs transfected with miR-342-3p inhibitor (G), or B6 BMDMs transfected with miR-342-3p mimic (H) after Mtb infection. Data are shown as the mean ± SEM of n = 3 biological replicates. I, J. Cell death mechanisms of C3H BMDMs transfected with miR-342-5p mimic (I), or B6 BMDMs transfected with miR-342-5p inhibitor (J), followed by Mtb infection for 36 h. Data are shown as the mean ± SEM of n = 3 biological replicates. Data information: ANOVA followed by Bonferroni post hoc test was used for data analysis (A, B, E-J). *P < 0.05, **P < 0.01. Abbreviation: n.s., not significant. NC, negative control. Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. MiR-342-3p directly targets SOCS6 to regulate anti-Mtb immunity A. Relative expressions of miR-342-2p target genes were analyzed by qRT–PCR. Data are shown as the mean ± SEM of n = 3 biological replicates. B. Cell death mechanisms of RAW264.7 cells transfected with siRNA, followed by Mtb infection for 36 h. Representative data (from n = 3 biological replicates) are shown as the mean ± SEM of technical replicates. C, D. MiR-342-3p mimic or inhibitor was transfected to RAW264.7 macrophages. After 48 h, cells were collected for qRT–PCR (C, data are shown as the mean ± SEM of n = 3 biological replicates) and Western blotting (D, representative blots from n = 3 biological replicates are shown) to detect the relative levels of SOCS6. E. C3H BMDMs were transfected with miR-342-3p mimic or SOCS6-overexpressing lentivirus, followed by Mtb infection. Cell death mechanisms were analyzed, respectively. Representative data (from n = 3 biological replicates) are shown as the mean ± SEM of technical replicates. F. B6 BMDMs were transfected with miR-342-3p inhibitor or Socs6 siRNA, followed by Mtb infection. Cell death mechanisms were analyzed, respectively. Representative data (from n = 3 biological replicates) are shown as the mean ± SEM of technical replicates. G, H. SOCS6-overexpressing vector (G) or Socs6 siRNA (H) was mixed with polyethylenimine to form a complex, which was used to infect mice by tail vein injection (N/P ratio=8). Lungs were collected for transfection efficiency validation. Data are shown as the mean ± SEM of n = 3 biological replicates. I. Alveolar macrophages from mice treated with SOCS6-overexpressing vector or Socs6 siRNA were collected to analyze cell death mechanisms. Data are shown as the mean ± SEM of n = 3 biological replicates. J, K. Secretion of cytokines TNF-α, IL-1, IL-6, and CXCL15 in BMDMs obtained from Mir342−/− B6 and Mir342−/− B6 mice supplemented with Socs6 siRNA (J), or from Mir342+/+ C3H and Mir342+/+ C3H mice supplemented with SOCS6-overexpressing vector (K), was detected by ELISA after Mtb stimulation. Data are shown as the mean ± SEM of n = 3 biological replicates. Data information: ANOVA followed by Bonferroni post hoc test (A, C, I-K) and two-tailed Student t test (G, H) were used for data analysis. *P < 0.05, **P < 0.01. Abbreviations: n.s., not significant. NC, negative control. Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. SOCS6 has no effects on IFN-γ, caspase 2, and caspase 9 A. Phosphorylation states of STAT family members in response to Mtb stimulation (4 h) in Socs6+/+ or Socs6−/− BMDMs were examined by Western blotting. Representative blots from n = 3 biological replicates are shown. B. Phosphorylation states of STAT1 and STAT3 in response to Mtb stimulation (4 h) in Socs6+/+ BMDMs were examined by Western blotting. Fludarabine treatment concentration was 10 μM, and the treatment time was 24 h. Representative blots from n = 3 biological replicates are shown. C. Phosphorylation states of STAT1 in response to Mtb stimulation in Socs6+/+ BMDMs were examined by Western blotting. Representative blots from n = 3 biological replicates are shown. D. Intracellular localization of STAT1 in Mtb-stimulated Socs6+/+ and Socs6−/− BMDMs were detected by immunofluorescence. Representative images from n = 3 biological replicates are shown. Scale bar = 100 μm. E, F. Relative expressions of chemokines CCL5, CXCL10, ICAM1, and caspase 3, caspase 7, caspase 8 in Mtb-stimulated Socs6+/+ (E) or Socs6−/− (F) BMDMs were detected by Western blotting. Representative blots from n = 3 biological replicates are shown. G. ELISA was performed to detect the secretion of IFN-γ in Socs6+/+ and Socs6−/− BMDMs during Mtb stimulation. Data are shown as the mean ± SEM of n = 3 biological replicates. H, I. caspase 2, caspase 9 in Mtb-stimulated Socs6+/+ or Socs6−/− BMDMs were detected by qRT–PCR (H, data are shown as the mean ± SEM of n = 3 biological replicates) and Western blotting (I, representative blots from n = 3 biological replicates are shown). J, K. Caspase 8 in STAT1-suppressed Socs6−/− BMDMs were detected by qRT–PCR (J, data are shown as the mean ± SEM of n = 3 biological replicates) and Western blotting (K, representative blots from n = 3 biological replicates are shown). Fludarabine (10 μM) was used to treat Socs6−/− BMDMs for 24 h to specifically suppress the activation of STAT1. Data information: ANOVA followed by Bonferroni post hoc test (G, H, J) was used for data analysis. **P < 0.01. Abbreviation: n.s., not significant. Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. RIPK3 is critical for SOCS6-regulated cell death mechanisms A. Relative expressions of RIPK1 and RIPK3 were analyzed by Western blotting in Socs6+/+ BMDMs stimulated with Mtb for 0–24 h. Representative blots from n = 3 biological replicates are shown. B. Socs6+/+ BMDMs were stimulated with Mtb for 0–24 h, and cell lysates were collected and immunoprecipitated using an anti-RIPK1 antibody. The recruitment of caspase 8, RIPK3, FADD, and MLKL was analyzed by immunoblots. The lower panel represents the immunoblot analysis of whole cell lysates. Representative blots from n = 3 biological replicates are shown. C. Cell death mechanisms of Mtb-infected Socs6−/− BMDMs that were transfected with plasmids expressing Myc-RIPK3 or Myc-RIPK3 K51A mutant as indicated. Representative data (from n = 3 biological replicates) are shown as the mean ± SEM of technical replicates. D. Socs6+/+ BMDMs were transfected with Ripk3 siRNA or Mlkl siRNA for 24 h. Afterward, transfected cells were stimulated with Mtb for 12 h, and cell lysates were collected and immunoprecipitated using an anti-RIPK1 antibody. The recruitment of caspase 8, RIPK3, FADD, and MLKL was analyzed by immunoblot. The lower panel represents the immunoblot analysis of whole cell lysates. Representative blots from n = 3 biological replicates are shown. E–H. Cell viabilities (E, data are shown as the mean ± SEM of n = 3 biological replicates), cell death mechanisms [F, data are shown as the mean ± SEM of n = 3 biological replicates. G, representative data (from n = 3 biological replicates) are shown as the mean ± SEM of technical replicates], or Mtb growth rates (H, data are shown as the mean ± SEM of n = 3 biological replicates) of Mtb-infected Socs6+/+ BMDMs that were transfected with Ripk3 siRNA or Mlkl siRNA as indicated. Z-VAD treatment concentration was 20 μM, and the treatment time was 24 h. I. Cell death mechanisms of Socs6−/− BMDMs transfected with plasmids expressing Myc-MLKL and stimulated with Mtb for 36 h. Data are shown as the mean ± SEM of n = 3 biological replicates. J. Cell viabilities of Socs6−/− BMDMs transfected with plasmids expressing Myc-MLKL for 24 h and stimulated with Mtb or z-VAD for 24 h. Data are shown as the mean ± SEM of n = 3 biological replicates. K. Mtb growth rates of Socs6−/− BMDMs transfected with plasmids expressing Myc-MLKL and stimulated with Mtb for 0–120 h. Data are shown as the mean ± SEM of n = 3 biological replicates. Data information: ANOVA followed by Bonferroni post hoc test (E, F, H-K) was used for data analysis. *P < 0.05, **P < 0.01. Abbreviation: n.s., not significant. Source data are available online for this figure. Download figure Download PowerPoint MiR-342-3p enhances the production of inflammatory cytokines We generated a Mir342+/+ C3H mouse strain carrying the pBROAD3-miR-342 sequence and a Mir342−/− B6 mouse strain with knockout of the pre-miR-342 sequence (Appendix␣Fig S1A and B). Compared to the control group, the survival time of Mir342+/+ C3H mice infected with 400 CFU Mtb was significantly lengthened (Fig 2A), while the survival time of Mtb-infected Mir342−/− B6 mice was remarkably shortened (Fig 2B). We also observed a statistically significant difference in bacterial loads in the lungs, spleens, and livers of Mir342+/+ C3H mice and their wild-type littermates (C3H) (Fig 2C) as well as Mir342−/− B6 mice and their wild-type littermates (B6) (Fig 2D). Mir342−/− B6 mice developed massive necrotic lung lesions after intravenous infection (Fig 2E, Appendix Table S1). In contrast, Mtb-infected Mir342+/+ C3H mice did not develop any lung lesions and only displayed trace levels of inflammation (Fig 2F, Appendix␣Table S1). Next, we detected the expression of inflammatory cytokines secreted by BMDMs during Mtb infection. Compared with littermate controls, Mir342+/+ C3H mice secreted increased levels of TNF-α, IL-1, IL-6, and CXCL15 (also known as IL-8) (Fig 2G). Deletion of miR-342 in B6 mice resulted in disordered cytokine secretion (Fig 2H). Taken together, these data indicate that miR-342-3p plays an important role in the switch between necrosis and apoptosis in Mtb-infected BMDMs and has a profound effect on anti-tuberculosis immunity. Figure 2. MiR-342-3p enhances the production of inflammatory cytokines A, B. Survival of Mir342+/+ C3H mice and their wild-type littermate controls (C3H) (n = 10) (A), or Mir342−/−
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