Research ArticleINFECTIOUS DISEASE

Host sirtuin 1 regulates mycobacterial immunopathogenesis and represents a therapeutic target against tuberculosis

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Science Immunology  24 Mar 2017:
Vol. 2, Issue 9, eaaj1789
DOI: 10.1126/sciimmunol.aaj1789
  • Fig. 1 Mtb infection down-regulates SIRT1 expression.

    (A) SIRT1 mRNA was assessed by qRT-PCR in Mtb-infected THP-1 cells. SIRT1 expression normalized to GAPDH expression, relative to uninfected (UN) cells, is shown. (B) Western blot analysis of SIRT1 and GAPDH (control) of Mtb-infected THP-1 cells as in (A). Uncropped image is provided in fig. S12A. (C) SIRT1 mRNA expression in the lungs of Mtb-infected mice. d, day; UN, uninfected mice (n = 4). (D) SIRT1 mRNA expression in the lungs of macaques with active or latent TB. Fold change of SIRT1 expression in lesions versus normal lung tissue is shown (n = 10 to 12). (E) Immunostaining of lung tissue of a representative uninfected and Mtb-infected macaques (either BCG-vaccinated or unvaccinated). Red, SIRT1 staining; green, nuclear staining. NC, necrotic core of granuloma. Magnification, ×20. Scale bars, 100 μm. (F) Percentage of pulmonary cells expressing SIRT1 from (E) (n = 8 to 20). (G to J) Raw intensity values for SIRT1 mRNA expression in the data set of different cohorts, that is, UK 2010, South Africa 2010, South Africa 2014, and China 2014. Red bars in (G) to (J) indicate the median. Data in (A) to (C) are representative of three to four independent experiments, expressed as means ± SEM, and analyzed by two-tailed Student’s t test. Data in (D) and (F) to (I) are analyzed by Mann-Whitney U test. Data in (J) are analyzed by paired Wilcoxon signed-rank test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Exact P values are provided in table S7.

  • Fig. 2 SIRT1 activators enhance control of Mtb growth.

    (A) Mtb growth after 24 hours in scrambled control [wild-type (WT)] and SIRT1 knockdown (SIRT1−/−) THP-1 cells presented as fold change compared with uninfected cells. An average of three independent experiments is shown. (B) Mtb growth after treatment of THP-1 cells with vehicle control or 100 μM RES. (C) Mtb growth after 24 hours in THP-1 cells treated with different doses of SRT. (D) Mtb growth after treatment of THP-1 cells with 5 μM SRT. (E) Mtb growth after 24 hours in WT and SIRT1−/− THP-1 cells treated with 5 μM SRT or 100 μM RES. Data are presented as fold change relative to vehicle control. (F) Growth of Mtb MDR strains after 72 hours in THP-1 cells treated with 100 μM INH, 5 μM SRT, or 100 μM RES. The name of each strain is indicated. Growth restriction by SRT and RES is significant over control by ANOVA. (G) Scatterplot of differentially expressed autophagy genes in Mtb-infected THP-1 cells (I) versus uninfected cells (U), and Mtb-infected THP-1 cells treated with RES (R) versus untreated infected cells (I). Positive and negative regulators of autophagy are enriched in upper left and lower right quadrants, respectively. lfc, log fold change. (H) Mtb-infected (IN) WT or SIRT1−/− THP-1 cells were treated with 5 μM SRT, 100 μM RES, or 2.5 μM rapamycin (Rapa) (positive control) for 4 hours, stained for LC3, and analyzed by flow cytometry. Representative histogram plot of five independent experiments is shown. Gray histogram, untreated cells. Quantification data of LC3B MFI (mean fluorescence intensity) are presented. (I) WT (DT), WT, or SIRT1−/− THP-1 cells were infected with Mtb and treated with 5 μM SRT for 24 hours. Cell lysates were subjected to immunoblot analysis for LC3 and GAPDH. LC3 II/LC3 I ratio is indicated. Uncropped image is provided in fig. S12B. (J) BCG growth after 24 hours in THP-1 cells treated or not with 5 μM SRT and 3-methyladenine (3-MA; 10 mM) or wortmannin (Wt; 5 μM). (K) THP-1 cells were infected with BCG-GFP and incubated with or without 5 μM SRT, 1 μM EX527 (EX) and EX + SRT, and 500 nM LTR for 4 hours before fixation. Scale bars, 5 μm. Zoomed view of boxed section is presented in the last column. (L) Quantification of LTR-positive BCG-GFP as in (K). Data in (B) to (E) and (I) to (L) are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-tailed Student’s t test. Exact P values are provided in table S8.

  • Fig. 3 SIRT1 activators normalize Mtb-induced inflammatory responses.

    (A) Circos figure depicting GO pathways modulated in Mtb-infected THP-1 cells treated or not with RES (24 hours). Bars in the gray circle represent the number of genes in each GO category. Bars in the yellow circle represent the number of genes in particular GO category that were significantly changed upon infection [I (infected) versus U (uninfected)]. Bars in the blue circle show the number of genes significantly modulated by RES treatment in the infected cells [R (RES-treated, infected) versus I (infected)]. A cutoff of 1 × 10−4 was used for the GO pathways. Black lines indicate pathways sharing at least 50% of genes. The thickness of the line shows the extent of overlaps between the two ontologies. (B) Comparison of 149 genes from inflammatory response (GO:0006954) shown in (A), present within 3062 genes that are modulated by RES (see fig. S4, A and B). Heat map displays absolute expression values of the up-regulated (yellow) and down-regulated (blue) genes in the respective analysis. (C) mRNA expression of IL6, TNFα, MCP-1, and IL1B in Mtb-infected THP-1 cells, treated or not with 5 μM SRT over a period of 72 hours. (D) Estimation of IL-1β, IL-6, and MCP-1 in the culture supernatant of Mtb-infected THP-1 cells at 24 hours after infection, treated with 5 μM SRT or control [dimethyl sulfoxide (DMSO)], measured by ELISA. (E) mRNA expression of RelA/p65 as in (C). (F) Immunoblots of cell lysates from uninfected (UN), lipopolysaccharide (LPS; 100 ng/ml)–stimulated, and Mtb-infected THP-1 cells, treated or not with 5 μM SRT over a period of 72 hours. Uncropped image is provided in fig. S12C. (G) Relative protein band density of RelA/p65 and acetylated RelA/p65 normalized to GAPDH at 72 hours. (H) Effect of SRT treatment on RelA/p65 recruitment to TNFα, IL1B, IL6, and MCP-1 promoters in Mtb-infected THP-1 cells. Aliquots of chromatin were obtained before (input) or after immunoprecipitation. Isolated chromatin was quantified by real-time PCR. Data are expressed as the fold change over the levels detected in the uninfected cells after correcting for differences in the amount of starting (input) chromatin material. Representative data from three (C to E) and two (F to H) independent experiments are shown. Data are means ± SEM performed in triplicate. *P < 0.05, **P < 0.01 by two-tailed Student’s t test. Exact P values are provided in table S9.

  • Fig. 4 SIRT1 activation reduces Mtb growth in mice.

    (A) Bacillary load in the lungs of control (WT) and Mac-SIRT1 KO mice infected with Mtb on day 21 after infection. Bar indicates the median. (B) Mtb-infected mice were treated with RES (50 or 100 mg/kg) starting 7 days after infection. Bacillary loads were enumerated in the lungs and spleen on days 1, 7, 21, and 35 after infection. Control, untreated infected mice. (C) Mtb-infected mice were treated with SRT (100 mg/kg) starting 7 days after infection. Bacillary loads were enumerated in the lungs and spleen on days 1, 7, 21, and 35 after infection. (D) Mtb-infected mice were treated with SRT (100 mg/kg) starting 40 days after infection. Bacillary loads in the lungs were enumerated on days 1, 7, 40, 55, and 68 after infection. n = 8 to 12 mice per group per time point. Data are means ± SEM. (A to D) Combined data of two independent experiments are shown. *P < 0.05, **P < 0.01 by Mann-Whitney U test. Exact P values are provided in table S10.

  • Fig. 5 SIRT1 activation reduces TB-associated tissue pathology and inflammation in mice.

    (A) Light micrographs of H&E staining of representative lung sections on day 35 after infection from Mtb-infected mice, treated or not with RES (100 mg/kg). Scale bars, 500 μm (×4 magnification) and 50 μm (×40 magnification). IN, Mtb-infected mice; IN + RES, RES-treated, Mtb-infected mice. (B) Percentage of lung area involved in disease pathology in IN and IN + RES mice. (C) Percentage of lung area involved in disease pathology of IN mice treated or not with SRT (100 mg/kg) on day 35 after infection. IN + SRT, SRT-treated, Mtb-infected mice. (D) mRNA expression was assessed by qRT-PCR in the lung RNA from IN and IN + SRT mice on day 35 after infection. (E) Absolute values of the inflammatory chemokines and cytokines from the lungs of Mtb-infected mice as in (D), measured by Luminex. (F) WT (control) and Mac-SIRT1 KO mice were infected with Mtb. On day 14, lung mRNA expression was assessed by qRT-PCR. (G) Estimation of IL-1β, TNFα, and MCP-1 in the lung homogenate of mice in (F) as measured by ELISA. Bar lines in (B) and (C) represent the median. Data in (D) to (G) are representative of two independent experiments. Data are means ± SEM. *P < 0.05, **P < 0.01 by Mann-Whitney U test. Exact P values are provided in table S11.

  • Fig. 6 SIRT1 activation modulates the lung myeloid landscape in Mtb-infected mice.

    (A) tSNE analysis of single-cell data from lung tissues of analyzed mice. Cells were plotted and color-coded by the 28 “unsupervised” DensVM clusters. A grouped description of each cluster is indicated. Detailed characterization of each cluster is shown in fig. S8. IMs, interstitial macrophages; dim, dimension; pDC, plasmacytoid DC; AM, alveolar macrophage. (B) Heat plot summary of normalized cluster frequency (unsupervised clustering) across the different groups of mice, treated or not with SRT (100 mg/kg). UN, uninfected mice; UN + SRT, uninfected mice treated with SRT; IN, Mtb-infected mice; IN + SRT, Mtb-infected mice treated with SRT. *, clusters that were significantly altered in IN versus UN; +, clusters that were significantly altered in IN + SRT versus IN. (C) Mass cytometry data were analyzed by manual gating strategy. MQs, macrophages. (D) tSNE analysis of cellular composition of lung tissues derived from UN, IN, and IN + SRT mice, highlighting six SRT modulated clusters. Each plot represents a pool of three to four lung tissue cells. Ungrouped cells (remaining) are shown in gray. (E) Mean frequencies of Ly6C subsets among CD45+ myeloid cells. Mass cytometry data were analyzed by manual gating strategy. (F and G) Fluorescent flow cytometric analysis of lung cells from SRT-treated, Mtb-infected animals. PerCP, peridinin chlorophyll; PE/Cy7, phycoerythrin/cyanin 7. (H) Number of lung monocytes in WT and Mac-SIRT1 KO mice on day 3 after infection. Data are means ± SEM. n = 4 to 6 mice per group per time point. *P < 0.05, **P < 0.01, #P < 0.005 by Mann-Whitney U test. Exact P values are provided in tables S12 and S13. FACS, fluorescence-activated cell sorting.

  • Table 1 SRT treatment enhances efficacy of INH in Mtb-infected mice.
    Mouse modelLungsSpleen
    INH*SRT + INHINHSRT + INH
    Acute
      Experiment 12/4 (50%)§4/5 (80%)4/5 (80%)5/5 (100%)
      Experiment 21/4 (25%)1/4 (25%)0/5 (0%)5/5 (100%)
    Chronic10.7 ± 2.5 × 104.9 ± 2.8 × 104Not performedNot performed

    *Mtb-infected mice were administered with INH (10 mg/kg).

    Mtb-infected mice were coadministered with INH and SRT (100 mg/kg).

    ‡Acute TB model proportion of mice with no detectable CFU in the lungs and spleen during INH therapy with and without SRT, after 4 weeks of treatment in two different experiments.

    §Number of mice with no detectable CFU out of total number of animals in that group (in bracket, percentage information is provided).

    ║In a chronic TB model bacterial load (CFU) in the lung of INH or SRT + INH–treated mice after 2 weeks of treatment.

    ¶Mean CFU values (n = 5). Difference in CFU values between INH and SRT + INH group is significant (P = 0.0317 by Mann-Whitney U test).

    Supplementary Materials

    • immunology.sciencemag.org/cgi/content/full/2/9/eaaj1789/DC1

      Methods

      Fig. S1. SIRT1 expression in Mtb-infected human cells and in the peripheral blood of humans infected with Mtb and other diseases.

      Fig. S2. SIRT1 is expressed by CD68/CD163 macrophages in granulomas of Mtb-infected macaques.

      Fig. S3. Down-regulation of SIRT1 expression by mycobacteria and control of mycobacterial growth by RES and SRT.

      Fig. S4. RES modulates the global gene expression during Mtb infection.

      Fig. S5. SIRT1 activation induces LC3 expression and phagosome-lysosome fusion in mycobacteria-infected THP-1 cells.

      Fig. S6. SRT reduces tissue Mtb load and Mtb-derived lung pathology, and SIRT1 deficient mouse has enhanced inflammatory response.

      Fig. S7. Examples of staining for each antibody used for the mass cytometry analysis.

      Fig. S8. Heat plot summary of average median expression of each cellular marker analyzed for the 28 clusters identified and rough descriptions of each cluster.

      Fig. S9. Manual gating strategy used to analyze mass cytometry and flow cytometry data.

      Fig. S10. Validation of tSNE-guided lung populations.

      Fig. S11. Lung myeloid cell population changes upon Mtb infection and SRT treatment.

      Fig. S12. Uncropped images of the Western blots.

      Table S1. TB data sets used in this study.

      Table S2. Differentially expressed genes between Mtb-infected THP-1 cells treated with RES and untreated cells.

      Table S3. Enrichment of significant GOs in 3062 differentially regulated genes between RES-treated, Mtb-infected THP-1 cells (R) and untreated, Mtb-infected THP-1 cells (I).

      Table S4. Enrichment of significant canonical pathways (IPA analysis) in differentially regulated genes between RES-treated, Mtb-infected THP-1 cells (R) and untreated, Mtb-infected THP-1 cells (I).

      Table S5. Antibodies used for mass cytometry analysis listing metal conjugate, antibody clone name, and supplier of each marker.

      Table S6. Sequences of primers used in this study.

      Table S7. P values for Fig. 1.

      Table S8. P values for Fig. 2.

      Table S9. P values for Fig. 3.

      Table S10. P values for Fig. 4.

      Table S11. P values for Fig. 5.

      Table S12. P values for Fig. 6B.

      Table S13. P values for Fig. 6 (C to H).

    • Supplementary Materials

      Supplementary Material for:

      Host sirtuin 1 regulates mycobacterial immunopathogenesis and represents a therapeutic target against tuberculosis

      Catherine Y. Cheng, Nuria M. Gutierrez, Mardiana B. Marzuki, Xiaohua Lu, Taylor W. Foreman, Bhairav Paleja, Bernett Lee, Akhila Balachander, Jinmiao Chen, Liana Tsenova, Natalia Kurepina, Karen W. W. Teng, Kim West, Smriti Mehra, Francesca Zolezzi, Michael Poidinger, Barry Kreiswirth, Deepak Kaushal, Hardy Kornfeld, Evan W. Newell, Amit Singhal*

      *Corresponding author. Email: amit_singhal{at}immunol.a-star.edu.sg

      Published 24 March 2017, Sci. Immunol. 2, eaaj1789 (2017)
      DOI: 10.1126/sciimmunol.aaj1789

      This PDF file includes:

      • Methods
      • Fig. S1. SIRT1 expression in Mtb-infected human cells and in the peripheral blood of humans infected with Mtb and other diseases.
      • Fig. S2. SIRT1 is expressed by CD68/CD163 macrophages in granulomas of Mtbinfected macaques.
      • Fig. S3. Down-regulation of SIRT1 expression by mycobacteria and control of mycobacterial growth by RES and SRT.
      • Fig. S4. RES modulates the global gene expression during Mtb infection.
      • Fig. S5. SIRT1 activation induces LC3 expression and phagosome-lysosome fusion in mycobacteria-infected THP-1 cells.
      • Fig. S6. SRT reduces tissue Mtb load and Mtb-derived lung pathology, and SIRT1 deficient mouse has enhanced inflammatory response.
      • Fig. S7. Examples of staining for each antibody used for the mass cytometry analysis.
      • Fig. S8. Heat plot summary of average median expression of each cellular marker analyzed for the 28 clusters identified and rough descriptions of each cluster.
      • Fig. S9. Manual gating strategy used to analyze mass cytometry and flow cytometry data.
      • Fig. S10. Validation of tSNE-guided lung populations.
      • Fig. S11. Lung myeloid cell population changes upon Mtb infection and SRT treatment.
      • Fig. S12. Uncropped images of the Western blots.
      • Table S1. TB data sets used in this study.
      • Table S3. Enrichment of significant GOs in 3062 differentially regulated genes between RES-treated, Mtb-infected THP-1 cells (R) and untreated, Mtb-infected THP-1 cells (I).
      • Table S5. Antibodies used for mass cytometry analysis listing metal conjugate, antibody clone name, and supplier of each marker.
      • Table S6. Sequences of primers used in this study.
      • Table S7. P values for Fig. 1.
      • Table S8. P values for Fig. 2.
      • Table S9. P values for Fig. 3.
      • Table S10. P values for Fig. 4.
      • Table S11. P values for Fig. 5.
      • Table S12. P values for Fig. 6B.
      • Table S13. P values for Fig. 6 (C to H).

      Download PDF

      Other Supplementary Material for this manuscript includes the following:

      • Table S2 (Microsoft Excel format). Differentially expressed genes between Mtb-infected THP-1 cells treated with RES and untreated cells.
      • Table S4 (Microsoft Excel format). Enrichment of significant canonical pathways (IPA analysis) in differentially regulated genes between RES-treated, Mtb-infected THP-1 cells (R) and untreated, Mtb-infected THP-1 cells (I).

      Files in this Data Supplement:

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