MiR-342 controls Mycobacterium tuberculosis susceptibility by modulating inflammation and cell death

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.

Keywords: apoptosis; inflammation; microRNA; tuberculosis; ubiquitination.

Figure 1. MiR‐342‐3p associates with TB susceptibility

1. 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.

2. 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.

3. 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.

4. 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.

5. 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.

Figure EV1. MiR‐342‐3p is associated with TB susceptibility

1. 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.

2. 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.

3. 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.

4. 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.

5. 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.

6. 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.

Figure EV2. MiR‐342‐3p directly targets SOCS6 to regulate anti‐Mtb immunity

1. 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.

2. 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.

3. 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.

4. 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.

5. 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.

6. 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.

7. 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.

8. 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.

Figure EV3. SOCS6 has no effects on IFN‐γ, caspase 2, and caspase 9

1. 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.

2. 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.

3. 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.

4. 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.

5. 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.

6. 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.

7. 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).

8. 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.

Figure EV4. RIPK3 is critical for SOCS6‐regulated cell death mechanisms

1. 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.

2. 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.

3. 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.

4. 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.

5. 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.

6. 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.

7. 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.

8. 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.

Figure 2. MiR‐342‐3p enhances the production of inflammatory cytokines

1. A, B

   Survival of Mir342 ^+/+ C3H mice and their wild‐type littermate controls (C3H) (n = 10) (A), or Mir342 ^−/− B6 mice and their wild‐type littermate controls (B6) (n = 10) (B) after aerosol infection with around 400 CFU Mtb. Data are shown as the Kaplan–Meier curves.

2. C, D

   Mtb bacterial loads in lungs, spleens, and livers of Mir342 ^+/+ C3H mice and their littermates (C3H) (C), or Mir342 ^−/− B6 mice and their littermates (B6) at 21 days post‐infection (dpi). Data are shown as the medians ± interquartile ranges of n = 3 biological replicates.

3. E, F

   Tuberculosis lung lesions of Mir342 ^−/− B6 mice and their littermates (B6) (E), or Mir342 ^+/+ C3H mice and their littermates (C3H) (F) were detected by hematoxylin/eosin staining at 21 dpi. Representative images from n = 3 biological replicates are shown. Scale bar = 100 μm.

4. G, H

   Secretion of cytokines TNF‐α, IL‐1, IL‐6, and CXCL15 in BMDMs obtained from Mir342 ^+/+ C3H and their littermates (C3H) (G), or from Mir342 ^−/− B6 and their littermates (B6) (H) after Mtb stimulation, was detected by ELISA. Data are shown as the mean ± SEM of n = 3 biological replicates.

Data information: Log‐rank (Mantel–Cox) test (A, B), Mann–Whitney U test (C, D), and ANOVA followed by Bonferroni post hoc test (G, H) were used for data analysis. *P < 0.05, **P < 0.01.

Figure 3. MicroRNA‐342‐3p directly targets SOCS6

1. A

   Predicted binding sites of miR‐342‐3p and Socs6 3′‐UTR.

2. B

   HEK‐293T fibroblasts were cotransfected with miR‐342‐3p mimic and luciferase reporter construct containing wild‐type or mutated UTR. After 24 h, cells were collected for luciferase assays. The Y‐axis showed relative luciferase activity. Data are shown as the mean ± SEM of n = 3 biological replicates.

3. C, D

   MiR‐342‐3p mimic or inhibitor was transfected to NIH‐3T3 fibroblasts. 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 SOCS6 expression.

4. E

   € RAW264.7 cells were transfected with Myc‐tagged Ago2 in the presence of miR‐342‐3p mimic or scramble for 24 h. Immunoprecipitation was performed with Myc antibody or IgG, and Socs6 mRNA was detected by qRT–PCR. Data are shown as the mean ± SEM of n = 3 biological replicates.

5. F–H

   C3H BMDMs were transfected with miR‐342‐3p mimic or SOCS6‐overexpressing lentivirus, followed by stimulation with Mtb or z‐VAD (20 μM) for 24 h. Cell death mechanisms (F), cell viabilities (G), and Mtb growth rates (H) were analyzed, respectively. Data are shown as the mean ± SEM of n = 3 biological replicates.

6. I–K

   B6 BMDMs were transfected with miR‐342‐3p inhibitor or Socs6 siRNA, followed by stimulation with Mtb or z‐VAD (20 μM) for 24 h. Cell death mechanisms (I), cell viabilities (J), and Mtb growth rates (K) were analyzed, respectively. Data are shown as the mean ± SEM of n = 3 biological replicates.

7. L

   Relative expressions of miR‐342‐3p and Socs6 in PBMCs from healthy individuals (n = 27) and TB patients (n = 34) were detected by qRT–PCR. The correlation between miR‐342‐3p and Socs6 was analyzed by Spearman test. Spearman rank correlation: r = −0.532, P < 0.001, n = 61.

Data information: ANOVA followed by Bonferroni post hoc test (B, C, E‐K) was 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.

Figure 4. SOCS6 participates in anti‐Mtb immunity

1. A, B

   Survival of Mir342 ^−/− B6 and Mir342 ^−/− B6 mice supplemented with Socs6 siRNA (n = 10) (A), or Mir342 ^+/+ C3H and Mir342 ^+/+ C3H mice supplemented with SOCS6‐overexpressing vector (n = 10) (B), after aerosol infection with around 400 CFU Mtb. Data are shown as the Kaplan–Meier curves.

2. C, D

   Mtb bacterial loads in lungs, spleens, and livers of Mir342 ^−/− B6 and Mir342 ^−/− B6 mice supplemented with Socs6 siRNA (C), or Mir342 ^+/+ C3H and Mir342 ^+/+ C3H mice supplemented with SOCS6‐overexpressing vector (D) at 21 dpi. Data are shown as the medians ± interquartile ranges of n = 3 biological replicates.

3. E, F

   Tuberculosis lung lesions of Mir342 ^−/− B6 and Mir342 ^−/− B6 mice supplemented with Socs6 siRNA (E), or Mir342 ^+/+ C3H and Mir342 ^+/+ C3H mice supplemented with SOCS6‐overexpressing vector (F) at 21 dpi were detected by hematoxylin/eosin staining. Representative images from n = 3 biological replicates are shown. Scale bar = 100 μm.

Data information: Log‐rank (Mantel–Cox) test (A, B) and Mann–Whitney U test (C, D) were used for data analysis. **P < 0.01.

Figure 5. SOCS6 regulates the expression of chemokines via modulating JAK/STAT signaling

1. A

   ELISA was performed to detect the secretion of cytokines TNF‐α, IL‐1, IL‐6, and CXCL15 in Socs6 ^+/+ and Socs6 ^−/− BMDMs during Mtb stimulation. Data are shown as the mean ± SEMof n = 3 biological replicates.

2. B

   Phosphorylation states of STAT1 in response to Mtb stimulation 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.

3. C–F

   Relative expressions of chemokines CCL5, CXCL10, ICAM1 (C), and caspase 3, caspase 7, caspase 8 (F) in Mtb‐stimulated Socs6 ^+/+ or Socs6 ^−/− BMDMs were detected by qRT–PCR. Data are shown as the mean ± SEM of n = 3 biological replicates. Cluster analysis of clinical samples. Heat maps showed relatively high (red) or low (green) expression levels of Socs6 and inflammatory factors in PBMCs from healthy individuals (n = 27) (D) and TB patients (n = 34) (E).

Data information: ANOVA followed by Bonferroni post hoc test (A, C, F) was used for data analysis. **P < 0.01. Abbreviation: n.s., not significant. Source data are available online for this figure.

Figure 6. SOCS6 determines cell death mechanisms via modulating recruitment of RIPK3

1. 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.

2. B

   Socs6^−/− BMDMs were stimulated with Mtb for 0–24 h, 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.

3. C

   Socs6^−/− BMDMs were transfected with plasmids expressing Myc‐RIPK3 or Myc‐RIPK3 K51A mutant 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.

4. D–F

   Cell viabilities (D), cell death mechanisms (E), or Mtb growth rates (F) of Mtb‐infected Socs6 ^−/− BMDMs that were transfected with plasmids expressing Myc‐RIPK3 or Myc‐RIPK3 K51A mutant as indicated. Z‐VAD treatment concentration was 20 μM and the treatment time was 24 h. Data are shown as the mean ± SEM of n = 3 biological replicates.

Data information: ANOVA followed by Bonferroni post hoc test (D‐F) was used for data analysis. *P < 0.05, **P < 0.01. Abbreviation: n.s., not significant. Source data are available online for this figure.

Figure 7. A20 controls degradation of RIPK3 through ubiquitin chains

1. A

   RAW264.7 cells were pretreated with or without E64d/PepA or MG132 for 4 h and then stimulated with or without Mtb for 12 h as indicated. Cell lysates were collected and immunoprecipitated using an anti‐RIPK3 antibody, and the ubiquitination of endogenous RIPK3 was detected using an anti‐Ub antibody. Representative blots from n = 3 biological replicates are shown.

2. B, C

   Socs6^+/+ BMDMs were transfected with miR‐342‐3p mimic or Socs6 siRNA (B), and Socs6 ^−/− BMDMs were transfected with plasmid expressing Myc‐SOCS6 (C) for 24 h as indicated. Transfected cells were then stimulated with Mtb for another 12 h, cell lysates were collected for immunoprecipitation, and ubiquitination of endogenous RIPK3 was detected by immunoblot. Representative blots from n = 3 biological replicates are shown.

3. D

   RAW264.7 cells were transfected with A20 siRNA, and cell lysates were collected for immunoprecipitation and immunoblot. Representative blots from n = 3 biological replicates are shown.

4. E–G

   RAW264.7 cells were transfected with plasmids expressing A20 and RIPK3 (E), or A20 and RIPK3 mutants (G) as indicated, cell lysates were collected for immunoprecipitation and immunoblot. Myc‐A20 or Flag‐RIPK3 purified from transfected HEK‐293T cells was incubated with ATP, E1, E2, and ubiquitin as indicated. The in␣vitro ubiquitination of RIPK3 was analyzed by immunoblot using an anti‐Ub antibody (F). Representative blots from n = 3 biological replicates are shown.

Source data are available online for this figure.

Figure 8. A20‐mediated cell death mechanism is independent of inflammatory responses

1. A, B

   Production of cytokines TNF‐α, IL‐1, IL‐6, CXCL15 (A), or chemokines Ccl5, Cxcl10, Icam1 (B) in the A20 ^−/− Socs6 ^+/+, A20 ^+/+ Socs6 ^+/+, A20 ^−/− Socs6 ^−/−, and A20 ^+/+ Socs6 ^−/− BMDMs after Mtb stimulation. Data are shown as the mean ± SEM of n = 3 biological replicates.

2. C–E

   Cell death mechanisms (C), cell viabilities (D), or Mtb growth rates (E) of A20 ^−/− Socs6 ^+/+, A20 ^+/+ Socs6 ^+/+, A20 ^−/− Socs6 ^−/−, and A20 ^+/+ Socs6 ^−/− BMDMs stimulated with Mtb. Z‐VAD treatment concentration was 20 μM, and the treatment time was 24 h. Data are shown as the mean ± SEM of n = 3 biological replicates.

3. F

   Schematic representation of miR‐342 regulated anti‐Mtb immunity.

Data information: ANOVA followed by Bonferroni post hoc test (A‐E) was used for data analysis. *P < 0.05, **P < 0.01. Abbreviation: n.s., not significant.