Research ArticleImmunology

Nuclear PTEN enhances the maturation of a microRNA regulon to limit MyD88-dependent susceptibility to sepsis

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Science Signaling  01 May 2018:
Vol. 11, Issue 528, eaai9085
DOI: 10.1126/scisignal.aai9085
  • Fig. 1 PTEN mRNA abundance is increased in murine sepsis and in the blood of septic pediatric and adult patients.

    (A) Quantitative polymerase chain reaction (qPCR) analysis of Pten mRNA expression in murine peritoneal cells 6 hours after CLP-induced sepsis or in sham-treated controls. Data are from n = 8 mice per group and were analyzed by t test and Mann-Whitney U test. (B) qPCR analysis of PTEN mRNA expression in the blood of adult septic patients who either survived (n = 18) or died (expired; n = 22). (C) PTEN mRNA abundance in the blood of normal control subjects (n = 52) and septic pediatric patients (n = 180) as determined by qPCR analysis. (D) PTEN mRNA abundance in the blood of pediatric septic patients who either developed comorbidities (endotype A; n = 60) or not (endotype B; n = 160) as determined by qPCR analysis. Data in (B) to (D) were analyzed by analysis of variance (ANOVA), and corrections for multiple comparisons were performed using a Benjamini-Hochberg false discovery rate of 5%. Data are expressed as relative abundance to normal control subjects. For the appropriate panels, *P < 0.05 compared to sham, surviving patients, normal controls, or endotype B.

  • Fig. 2 Myeloid PTEN inhibits SIRS and improves animal survival and bacterial clearance during sepsis.

    (A) Survival rates of C57BL/6 mice treated with scrambled control siRNA or PTEN-specific siRNA or treated with vehicle or the PTEN inhibitor Bpvic(OH) before being subjected to moderate CLP. Survival was monitored for 7 days. Data are from n = 10 mice per group and were analyzed by log-rank (Mantel-Cox) test. (B to D) The peritoneal exudates of mice treated with the indicated inhibitors or siRNAs were isolated 6 hours after CLP-induced sepsis. Bacterial burden (B) and neutrophil recruitment (C and D) were determined. Data are from n = 7 mice per group and were analyzed by one-way ANOVA, followed by Bonferroni correction. PMN, polymorphonuclear neutrophil. (E) Concentrations of IL-1β, TNF-α, and IL-6 in the peritoneal exudates of mice treated as described in (A). Data are from n = 7 mice per group and were analyzed by one-way ANOVA, followed by Bonferroni correction. (F) Survival rates of PTENfl/fl (control) and PTENfl/fl_lysMcre mice subjected to moderate CLP-induced sepsis. Survival was monitored for 7 days. Data are from n = 14 mice per group and were analyzed by log-rank (Mantel-Cox) test. Inset: The abundance of PTEN and actin (internal control) in peritoneal cells from PTENfl/fl and PTENfl/fl_lysMcre mice was determined by Western blotting analysis. (G) Production of TNF-α, IL-1β, and IL-6 in the serum and peritoneal cavity 6 hours after CLP-induced sepsis in PTENfl/fl and PTENfl/fl_lysMcre mice. Data are from n = 7 mice per group and were analyzed by one-way ANOVA, followed by Bonferroni correction. For the appropriate panels, *P < 0.05 compared to sham mice, vehicle, scrambled siRNA control, or PTENfl/fl mice. PTEN inhib, Bpvic(OH); PC, peritoneal cavity.

  • Fig. 3 PTEN inhibits MyD88 expression and prevents TLR2 and TLR4 activation in macrophages.

    (A) Isolated macrophages were transfected with scrambled or PTEN-specific siRNA. Forty-eight hours later, the cells were stimulated with the indicated TLR agonists for 30 min. Left: Cell lysates were analyzed by Western blotting with antibodies against the phosphorylated form of NF-κB p65 (p-p65), PTEN, and actin. Right: Quantification of the normalized p-p65 band intensity from at least three independent experiments. Data are means ± SEM. (B) Macrophages transfected as described in (A) were left unstimulated or stimulated with LPS for 24 hours, and the amount of nitrite produced in the cell culture medium was determined by Griess assay. Data are means ± SEM of at least three independent experiments. (C) Left: Macrophages were transfected as described in (A), and cell lysates were analyzed by Western blotting to detect the indicated proteins. t-PTEN, total PTEN protein. Right: Quantification of relative PTEN and MyD88 band intensities from at least three independent experiments. Data are means ± SEM. (D) WT mice were injected intraperitoneally with scrambled siRNA or PTEN-specific siRNA. The abundance of PTEN and MyD88 proteins in resident peritoneal cells was determined by Western blotting analysis. Data are from two mice per group. (E) Left: Macrophages from PTENfl/fl and PTENfl/fl_lysMcre mice were challenged with LPS for the indicated times, and MyD88 abundance in the cell lysates was determined by Western blotting analysis. Right: Quantification of relative MyD88 band intensities from at least three independent experiments. Data are means ± SEM. For the appropriate panels, *P < 0.05 compared to scrambled siRNA control group or PTENfl/fl; #P < 0.05 compared to LPS-stimulated PTEN siRNA or PTENfl/fl_lysMcre compared to scrambled siRNA control group or PTENfl/fl macrophages. In all circumstances, at least three independent experiments were performed, and data were analyzed by one-way ANOVA, followed by Bonferroni correction.

  • Fig. 4 Blocking MyD88 prevents enhanced mortality, bacterial load, and SIRS in siPTEN-challenged mice.

    (A) C57BL/6 mice were treated with PTEN inhibitor or vehicle control for 24 hours before being subjected to CLP-induced sepsis. Myd88 mRNA abundance was then determined in peritoneal cells 24 hours after sepsis by qPCR analysis. The abundance of Myd88 mRNA is expressed relative to that in sham-operated mice. Data are means ± SEM of n = 4 to 6 mice per group and were analyzed by one-way ANOVA, followed by Bonferroni correction. (B) WT mice were treated with scrambled siRNA control or PTEN-specific siRNA before undergoing CLP, which was followed by treatment with MyD88 peptide inhibitor for 1 hour. Survival was monitored for 7 days. Data are from n = 13 mice per group and were analyzed by log-rank (Mantel-Cox) test. (C and D) WT mice were treated as described in (B), and bacterial burden (C) and cytokine production (D) were determined in peritoneal exudates 24 hours after CLP. Data are means ± SEM of at least five mice per group and were analyzed by one-way ANOVA, followed by Bonferroni correction. For the appropriate panels, *P < 0.01 compared to sham, vehicle control, scrambled siRNA control, or PTENfl/fl; #P < 0.01 compared to PTEN siRNA group; &P < 0.05 compared to PTEN siRNA plus scrambled control. CFU, colony-forming unit.

  • Fig. 5 PTEN lipid phosphatase activity increases the abundance of miRNAs involved in regulating Myd88 expression and TLR activation in macrophages.

    (A) Elicited macrophages were transfected with scrambled siRNA control or PTEN-specific siRNA. Twenty-four hours later, miRNAs were isolated and an miRNA-focused array was performed. (B) Elicited macrophages were transfected with PTEN-specific siRNA as described in (A), and the abundances of the indicated miRNAs were determined by qPCR analysis. Data are expressed relative to the miRNA abundance in the scrambled control siRNA samples. Data are means ± SEM. (C) Elicited macrophages were transduced with PTEN-expressing adenovirus (Ad) or empty vector. Forty-eight hours later, the abundances of indicated miRNAs were determined by qPCR analysis. Abundance relative to that in cells transfected with empty vector adenovirus (EV-Ad) is expressed as means ± SEM. (D) Macrophages from PTENfl/fl and PTENfl/fl_lysMcre mice were isolated, and the indicated miRNAs were detected by qPCR. Abundance relative to that in PTENfl/fl macrophages is expressed as means ± SEM. (E) CLP-induced sepsis was performed in PTENfl/fl and PTENfl/fl_lysMcre mice, and miRNA abundance was measured in peritoneal cells 24 hours after CLP by qPCR analysis. Abundance relative to that in the control siRNA–treated sham mice is expressed as means ± SEM. (F) Macrophages from PTENfl/fl and PTENfl/fl_lysMcre mice were treated with the PI3K inhibitor wortmannin (Wortm) or the mTOR inhibitor rapamycin (Rapam) for 24 hours, and the abundance of miR125b was determined by qPCR. Abundance relative to that in PTENfl/fl macrophages treated with vehicle control was expressed as means ± SEM. (G) Elicited peritoneal macrophages from WT mice were transfected with PTEN-specific siRNA or scrambled siRNA as described in (A) and with the indicated miRNA mimics. Twenty-four hours later, Myd88 mRNA abundance was determined by qPCR analysis. Abundance relative to that in macrophages treated with scrambled control siRNA and the mimic control is expressed as means ± SEM. (H) Macrophages from C57BL/6 mice were treated as described in (G) and challenged with LPS for 24 hours. Nitrite concentrations in the cell culture medium were determined by Griess assay. Data are means ± SEM of at least five mice per group repeated at three independent times and were analyzed by one-way ANOVA, followed by Bonferroni correction. *P < 0.05 compared to the scrambled siRNA control or PTENfl/fl mice; #P < 0.05 for LPS-stimulated PTEN siRNA compared to scrambled siRNA control or PTENfl/fl macrophages; &P < 0.05 compared to miRNA mimic alone.

  • Fig. 6 Nuclear PTEN drives miRNA processing.

    (A) PTEN−/− MEFs were transduced with retrovirus-containing empty vector control (TRE) or the indicated WT or mutant PTEN constructs. Expression of the indicated miRNAs was determined by qPCR analysis. Abundance relative to that in untransfected cells (TRE) was expressed as means ± SEM of n = 3 independent experiments. Data were analyzed by one-way ANOVA, followed by Bonferroni correction. (B) Macrophages were isolated from PTENfl/fl and PTENfl/fl_lysMcre mice, and the abundances of Drosha, Dgcr8, Xpo5, and Dicer 1 mRNAs were determined by qPCR analysis. Abundance relative to that in macrophages from PTENfl/fl mice was expressed as means ± SEM of n = 4 mice per group. (C) The abundances of the indicated primary miRNAs in macrophages from PTENfl/fl and PTENfl/fl_lysMcre mice were determined by qPCR analysis. Abundance relative to that in macrophages from PTENfl/fl mice was expressed as means ± SEM of n = 4 mice per group. (D) Macrophages were treated with scrambled control or PTEN-specific siRNA, and the abundances of the indicated primary miRNAs were determined by qPCR analysis. Abundance relative to that in macrophages treated with control siRNA was expressed as means ± SEM of n = 5 mice per group. (E) Left: Macrophages from C57BL/6 mice were incubated with antibodies recognizing Drosha, Dgcr8, Dicer, or PTEN, as indicated, and visualized by confocal microscopy. Right: Overlap coefficients between PTEN/DICER, PTEN/Drosha, and PTEN/Dgcr8. Data are means ± SEM of three independent experiments, with ~100 cells analyzed in each experimental group. 4′,6-Diamidino-2-phenylindole (DAPI) staining is in blue. Each field is representative of 100 cells examined (original magnification, ×400) from each of three independent experiments with the values of the DICER/PTEN association set as 1. (F) Left: In situ PLA of PTEN/Drosha or PTEN/Dgcr8 complexes in elicited macrophages from PTENfl/fl and PTENfl/fl_lysMcre mice. PLA complexes are shown in red, and nuclei are shown in blue. Right: Numbers of specks/nucleus in at least 100 cells expressed as means ± SEM of at least three independent experiments. (G) Left: PTEN was immunoprecipitated (IP) from total macrophage cell lysates from PTENfl/fl and PTENfl/fl_lysMcre mice, and samples were then analyzed by Western blotting with antibodies against the indicated proteins. Right: The relative densities of bands corresponding to PTEN, Dgcr8, Drosha, and DICER were determined by densitometry analysis. Data are means ± SEM of three individual experiments, with the values of the PTENfl/fl control group set as 1. IB, immunoblotting. (H) Left: Macrophages from PTENfl/fl and PTENfl/fl_lysMcre mice were incubated with antibodies recognizing Drosha, Dgcr8, or Dicer, as indicated, and visualized by confocal microcopy. Confocal images were captured under identical settings to enable comparison of staining intensities. Images are from one experiment, which are representative of five independent experiments. Right: Overlap coefficients between DAPI (nuclear, blue) and Dgcr8, Drosha, or DICER (red). Data are means ± SEM of three independent experiments, with ~100 cells analyzed in each experimental group. Values from PTENfl/fl cells were set to 1. (I) Left: Nuclear and cytosolic fractions of macrophage lysates from PTENfl/fl and PTENfl/fl_lysMcre mice were isolated and analyzed by Western blotting with antibodies against the indicated proteins. Right: The relative intensities of bands corresponding to DGCR8, Drosha, and histone H3 were determined by densitometry analysis. Data are means ± SEM of three individual experiments, with the values of the cytosolic proteins from the PTENfl/fl control group set as 1. For the appropriate panels, *P < 0.05 compared to empty vector, control scrambled siRNA, or PTENfl/fl mice.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/528/eaai9085/DC1

    Fig. S1. Antibiotic does not prevent PTEN inhibition–induced animal lethality during sepsis.

    Fig. S2. PTEN controls MRSA-induced peritonitis.

    Fig. S3. PTEN protects mice against lung injury during sepsis.

    Fig. S4. PTEN does not control TRIF-dependent macrophage activation.

    Fig. S5. PTEN does not dephosphorylate IRAK4 or IKKα in macrophage lysates.

    Fig. S6. PTEN lipid phosphatase activity decreases MyD88 mRNA and protein abundance in alveolar macrophages.

    Fig. S7. MyD88-blocking peptide prevents TLR4- and TLR2-mediated, but not TLR3-mediated, nitrite production.

    Fig. S8. PTEN does not target transcription factors involved in basal Myd88 expression in macrophages.

    Fig. S9. PTEN controls miRNA abundance in alveolar macrophages.

    Fig. S10. Differential roles of mTOR and PI3K in miRNA expression.

    Fig. S11. The miRNAs miR125b and miR203 directly reduce Myd88 mRNA abundance in macrophages.

    Fig. S12. Efficiency of PTEN-expressing retrovirus in PTEN−/− MEFs.

    Table S1. Adult sepsis patient demographics.

  • Supplementary Materials for:

    Nuclear PTEN enhances the maturation of a microRNA regulon to limit MyD88-dependent susceptibility to sepsis

    Flavia Sisti, Soujuan Wang, Stephanie L. Brandt, Nicole Glosson-Byers, Lindsey D. Mayo, Young Min Son, Sarah Sturgeon, Luciano Filgueiras, Sonia Jancar, Hector Wong, Charles S. Dela Cruz, Nathaniel Andrews, Jose Carlos Alves-Filho, Fernando Q. Cunha, C. Henrique Serezani*

    *Corresponding author. Email: h.serezan{at}vanderbilt.edu

    This PDF file includes:

    • Fig. S1. Antibiotic does not prevent PTEN inhibition–induced animal lethality during sepsis.
    • Fig. S2. PTEN controls MRSA-induced peritonitis.
    • Fig. S3. PTEN protects mice against lung injury during sepsis.
    • Fig. S4. PTEN does not control TRIF-dependent macrophage activation.
    • Fig. S5. PTEN does not dephosphorylate IRAK4 or IKKα in macrophage lysates.
    • Fig. S6. PTEN lipid phosphatase activity decreases MyD88 mRNA and protein abundance in alveolar macrophages.
    • Fig. S7. MyD88-blocking peptide prevents TLR4- and TLR2-mediated, but not TLR3-mediated, nitrite production.
    • Fig. S8. PTEN does not target transcription factors involved in basal Myd88 expression in macrophages.
    • Fig. S9. PTEN controls miRNA abundance in alveolar macrophages.
    • Fig. S10. Differential roles of mTOR and PI3K in miRNA expression.
    • Fig. S11. The miRNAs miR125b and miR203 directly reduce Myd88 mRNA abundance in macrophages.
    • Fig. S12. Efficiency of PTEN-expressing retrovirus in PTEN−/− MEFs.
    • Table S1. Adult sepsis patient demographics.

    [Download PDF]


    © 2018 American Association for the Advancement of Science

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