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Fine-tuning of macrophage activation using synthetic rocaglate derivatives.

Bhattacharya B, Chatterjee S, Devine WG, Kobzik L, Beeler AB, Porco JA, Kramnik I - Sci Rep (2016)

Bottom Line: Several active compounds belonged to the flavagline (rocaglate) family.These compounds may represent a basis for macrophage-directed therapies that fine-tune macrophage effector functions to combat intracellular pathogens and reduce inflammatory tissue damage.These therapies would be especially relevant to fighting drug-resistant pathogens, where improving host immunity may prove to be the ultimate resource.

View Article: PubMed Central - PubMed

Affiliation: Pulmonary Center, Department of Medicine, Boston University School of Medicine, National Emerging Infectious Diseases Laboratories (NEIDL), Boston University, Boston, MA, 02118, USA.

ABSTRACT
Drug-resistant bacteria represent a significant global threat. Given the dearth of new antibiotics, host-directed therapies (HDTs) are especially desirable. As IFN-gamma (IFNγ) plays a central role in host resistance to intracellular bacteria, including Mycobacterium tuberculosis, we searched for small molecules to augment the IFNγ response in macrophages. Using an interferon-inducible nuclear protein Ipr1 as a biomarker of macrophage activation, we performed a high-throughput screen and identified molecules that synergized with low concentration of IFNγ. Several active compounds belonged to the flavagline (rocaglate) family. In primary macrophages a subset of rocaglates 1) synergized with low concentrations of IFNγ in stimulating expression of a subset of IFN-inducible genes, including a key regulator of the IFNγ network, Irf1; 2) suppressed the expression of inducible nitric oxide synthase and type I IFN and 3) induced autophagy. These compounds may represent a basis for macrophage-directed therapies that fine-tune macrophage effector functions to combat intracellular pathogens and reduce inflammatory tissue damage. These therapies would be especially relevant to fighting drug-resistant pathogens, where improving host immunity may prove to be the ultimate resource.

No MeSH data available.


Related in: MedlinePlus

C9433 induces autophagy in primary macrophages and promotes bacterial clearance.(a) BMDM were treated with different doses of C9433 for 16 hrs and probed with LC3B mAb. A mix of chloroquine(10 μM) and rapamycin(500 nM) was used as a positive control (C). (b) BMDM was treated with compound for 16 hrs and autophagy induction was determined using Cyto-ID Autophagy Detection kit. (c) BMDM were treated with 1 μM compound for indicated time and probed with LC3B mAb. (d) BMDM were treated with 1 μM compound for 6 hrs. Bafilomycin(50 nM) and chloroquine(30 μM) were added to the cells 2 hrs before harvesting and probed with LC3B antibody. All blots represent data from two independent experiments. (e) iBMM cells were treated with 1 μM compound for 6 hrs and autophagic puncta was detected using confocal microscopy. (f) BMDM were treated with 15 ng/mL TNFα and 10 U/mL IFNγ for 24 hrs in the presence and absence of C9433 and NO production was determined. (g) iNOS expression was determined in the above samples using specific anti-iNOS antibody using celigo cytometer. Two independent experiments performed in triplicates. (h) BMDM were treated with 10 ng/mL TNFα in the presence of C9433 for 24 hrs. mRNA expression of IFNβ, IP10 and IL10 were measured by q-PCR. Data is represented as % of gene expression relative to 10 ng/ml TNFα treated cells from two independent experiments. (i) BMDMs were either untreated or pretreated with 2 μM C9433, 0.5 U/mL of IFNγ alone or in presence of compound for 16 hrs and then infected with F.t. LVS at MOI 1 for 24 hrs. Bacteria were detected using anti-F.t. LVS antibodies(red), nuclei counterstained with DAPI(blue). (j) 100 cells were counted per condition to detect intracellular bacteria and the % of infected cells were calculated. Microscope images represent data from at least two independent experiments. (k) F.t. LVS infected BMDM in presence and absence of compound were probed with anti-LC3B mAb. (l) BMDM were pretreated with C9433 for 16 hrs in the presence or absence of IFNγ and then infected with F.t. LVS at MOI 10. After 7 hrs% of PI- positive cells were enumerated using celigo cytometer. Two independent experiments were performed in triplicates.
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f4: C9433 induces autophagy in primary macrophages and promotes bacterial clearance.(a) BMDM were treated with different doses of C9433 for 16 hrs and probed with LC3B mAb. A mix of chloroquine(10 μM) and rapamycin(500 nM) was used as a positive control (C). (b) BMDM was treated with compound for 16 hrs and autophagy induction was determined using Cyto-ID Autophagy Detection kit. (c) BMDM were treated with 1 μM compound for indicated time and probed with LC3B mAb. (d) BMDM were treated with 1 μM compound for 6 hrs. Bafilomycin(50 nM) and chloroquine(30 μM) were added to the cells 2 hrs before harvesting and probed with LC3B antibody. All blots represent data from two independent experiments. (e) iBMM cells were treated with 1 μM compound for 6 hrs and autophagic puncta was detected using confocal microscopy. (f) BMDM were treated with 15 ng/mL TNFα and 10 U/mL IFNγ for 24 hrs in the presence and absence of C9433 and NO production was determined. (g) iNOS expression was determined in the above samples using specific anti-iNOS antibody using celigo cytometer. Two independent experiments performed in triplicates. (h) BMDM were treated with 10 ng/mL TNFα in the presence of C9433 for 24 hrs. mRNA expression of IFNβ, IP10 and IL10 were measured by q-PCR. Data is represented as % of gene expression relative to 10 ng/ml TNFα treated cells from two independent experiments. (i) BMDMs were either untreated or pretreated with 2 μM C9433, 0.5 U/mL of IFNγ alone or in presence of compound for 16 hrs and then infected with F.t. LVS at MOI 1 for 24 hrs. Bacteria were detected using anti-F.t. LVS antibodies(red), nuclei counterstained with DAPI(blue). (j) 100 cells were counted per condition to detect intracellular bacteria and the % of infected cells were calculated. Microscope images represent data from at least two independent experiments. (k) F.t. LVS infected BMDM in presence and absence of compound were probed with anti-LC3B mAb. (l) BMDM were pretreated with C9433 for 16 hrs in the presence or absence of IFNγ and then infected with F.t. LVS at MOI 10. After 7 hrs% of PI- positive cells were enumerated using celigo cytometer. Two independent experiments were performed in triplicates.

Mentions: In GSEA analysis, the highest positive normalized enrichment score (1.81) was for genes that contained a NF-κB element in their promoters, suggesting that C9433 in combination with IFNγ enhanced NF-κB activity. The expression of NF-κB -inducing kinase (NIK, Map3K14) and TNFα were up-regulated 5-fold suggesting a potential mechanism for NF-κB activation. In addition, up-regulation of a number of stress response genes (ATF4, ATF6, Ddit3, Trib3, Gadd45b, Gadd45g, Hspa1a, and Osgin1) suggested that activation of stress responses might contribute to NF-κB activation by C9433 as well. NF-κB activation may explain superinduction of the Irf1 gene, since it contains a combinatorial NF-κB/GAS element in its promoter30. Several other interferon-inducible genes were up-regulated in C9433 plus IFNγ co-stimulated cells (irg1, ifi205, ifit1, ifrd1, igtp). Notably an interferon-inducible negative regulator of IFN-I pathway Usp18 was up-regulated 7.8-fold. We confirmed the C9433-mediated suppression of the IFN-I pathway in an independent assay, where macrophages were stimulated with TNFα (10 ng/mL), a treatment known to induce moderate IFN-I pathway activation31. We observed potent suppression of IFNβ, and its downstream targets IP-10 and IL-10 by C9433 (Fig. 4h). Perhaps, the direct suppressive effect of C9433 on the IFN-I pathway may partially account for the sensitization of macrophages to IFNγ,as the two pathways are known to antagonize each other32.


Fine-tuning of macrophage activation using synthetic rocaglate derivatives.

Bhattacharya B, Chatterjee S, Devine WG, Kobzik L, Beeler AB, Porco JA, Kramnik I - Sci Rep (2016)

C9433 induces autophagy in primary macrophages and promotes bacterial clearance.(a) BMDM were treated with different doses of C9433 for 16 hrs and probed with LC3B mAb. A mix of chloroquine(10 μM) and rapamycin(500 nM) was used as a positive control (C). (b) BMDM was treated with compound for 16 hrs and autophagy induction was determined using Cyto-ID Autophagy Detection kit. (c) BMDM were treated with 1 μM compound for indicated time and probed with LC3B mAb. (d) BMDM were treated with 1 μM compound for 6 hrs. Bafilomycin(50 nM) and chloroquine(30 μM) were added to the cells 2 hrs before harvesting and probed with LC3B antibody. All blots represent data from two independent experiments. (e) iBMM cells were treated with 1 μM compound for 6 hrs and autophagic puncta was detected using confocal microscopy. (f) BMDM were treated with 15 ng/mL TNFα and 10 U/mL IFNγ for 24 hrs in the presence and absence of C9433 and NO production was determined. (g) iNOS expression was determined in the above samples using specific anti-iNOS antibody using celigo cytometer. Two independent experiments performed in triplicates. (h) BMDM were treated with 10 ng/mL TNFα in the presence of C9433 for 24 hrs. mRNA expression of IFNβ, IP10 and IL10 were measured by q-PCR. Data is represented as % of gene expression relative to 10 ng/ml TNFα treated cells from two independent experiments. (i) BMDMs were either untreated or pretreated with 2 μM C9433, 0.5 U/mL of IFNγ alone or in presence of compound for 16 hrs and then infected with F.t. LVS at MOI 1 for 24 hrs. Bacteria were detected using anti-F.t. LVS antibodies(red), nuclei counterstained with DAPI(blue). (j) 100 cells were counted per condition to detect intracellular bacteria and the % of infected cells were calculated. Microscope images represent data from at least two independent experiments. (k) F.t. LVS infected BMDM in presence and absence of compound were probed with anti-LC3B mAb. (l) BMDM were pretreated with C9433 for 16 hrs in the presence or absence of IFNγ and then infected with F.t. LVS at MOI 10. After 7 hrs% of PI- positive cells were enumerated using celigo cytometer. Two independent experiments were performed in triplicates.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4834551&req=5

f4: C9433 induces autophagy in primary macrophages and promotes bacterial clearance.(a) BMDM were treated with different doses of C9433 for 16 hrs and probed with LC3B mAb. A mix of chloroquine(10 μM) and rapamycin(500 nM) was used as a positive control (C). (b) BMDM was treated with compound for 16 hrs and autophagy induction was determined using Cyto-ID Autophagy Detection kit. (c) BMDM were treated with 1 μM compound for indicated time and probed with LC3B mAb. (d) BMDM were treated with 1 μM compound for 6 hrs. Bafilomycin(50 nM) and chloroquine(30 μM) were added to the cells 2 hrs before harvesting and probed with LC3B antibody. All blots represent data from two independent experiments. (e) iBMM cells were treated with 1 μM compound for 6 hrs and autophagic puncta was detected using confocal microscopy. (f) BMDM were treated with 15 ng/mL TNFα and 10 U/mL IFNγ for 24 hrs in the presence and absence of C9433 and NO production was determined. (g) iNOS expression was determined in the above samples using specific anti-iNOS antibody using celigo cytometer. Two independent experiments performed in triplicates. (h) BMDM were treated with 10 ng/mL TNFα in the presence of C9433 for 24 hrs. mRNA expression of IFNβ, IP10 and IL10 were measured by q-PCR. Data is represented as % of gene expression relative to 10 ng/ml TNFα treated cells from two independent experiments. (i) BMDMs were either untreated or pretreated with 2 μM C9433, 0.5 U/mL of IFNγ alone or in presence of compound for 16 hrs and then infected with F.t. LVS at MOI 1 for 24 hrs. Bacteria were detected using anti-F.t. LVS antibodies(red), nuclei counterstained with DAPI(blue). (j) 100 cells were counted per condition to detect intracellular bacteria and the % of infected cells were calculated. Microscope images represent data from at least two independent experiments. (k) F.t. LVS infected BMDM in presence and absence of compound were probed with anti-LC3B mAb. (l) BMDM were pretreated with C9433 for 16 hrs in the presence or absence of IFNγ and then infected with F.t. LVS at MOI 10. After 7 hrs% of PI- positive cells were enumerated using celigo cytometer. Two independent experiments were performed in triplicates.
Mentions: In GSEA analysis, the highest positive normalized enrichment score (1.81) was for genes that contained a NF-κB element in their promoters, suggesting that C9433 in combination with IFNγ enhanced NF-κB activity. The expression of NF-κB -inducing kinase (NIK, Map3K14) and TNFα were up-regulated 5-fold suggesting a potential mechanism for NF-κB activation. In addition, up-regulation of a number of stress response genes (ATF4, ATF6, Ddit3, Trib3, Gadd45b, Gadd45g, Hspa1a, and Osgin1) suggested that activation of stress responses might contribute to NF-κB activation by C9433 as well. NF-κB activation may explain superinduction of the Irf1 gene, since it contains a combinatorial NF-κB/GAS element in its promoter30. Several other interferon-inducible genes were up-regulated in C9433 plus IFNγ co-stimulated cells (irg1, ifi205, ifit1, ifrd1, igtp). Notably an interferon-inducible negative regulator of IFN-I pathway Usp18 was up-regulated 7.8-fold. We confirmed the C9433-mediated suppression of the IFN-I pathway in an independent assay, where macrophages were stimulated with TNFα (10 ng/mL), a treatment known to induce moderate IFN-I pathway activation31. We observed potent suppression of IFNβ, and its downstream targets IP-10 and IL-10 by C9433 (Fig. 4h). Perhaps, the direct suppressive effect of C9433 on the IFN-I pathway may partially account for the sensitization of macrophages to IFNγ,as the two pathways are known to antagonize each other32.

Bottom Line: Several active compounds belonged to the flavagline (rocaglate) family.These compounds may represent a basis for macrophage-directed therapies that fine-tune macrophage effector functions to combat intracellular pathogens and reduce inflammatory tissue damage.These therapies would be especially relevant to fighting drug-resistant pathogens, where improving host immunity may prove to be the ultimate resource.

View Article: PubMed Central - PubMed

Affiliation: Pulmonary Center, Department of Medicine, Boston University School of Medicine, National Emerging Infectious Diseases Laboratories (NEIDL), Boston University, Boston, MA, 02118, USA.

ABSTRACT
Drug-resistant bacteria represent a significant global threat. Given the dearth of new antibiotics, host-directed therapies (HDTs) are especially desirable. As IFN-gamma (IFNγ) plays a central role in host resistance to intracellular bacteria, including Mycobacterium tuberculosis, we searched for small molecules to augment the IFNγ response in macrophages. Using an interferon-inducible nuclear protein Ipr1 as a biomarker of macrophage activation, we performed a high-throughput screen and identified molecules that synergized with low concentration of IFNγ. Several active compounds belonged to the flavagline (rocaglate) family. In primary macrophages a subset of rocaglates 1) synergized with low concentrations of IFNγ in stimulating expression of a subset of IFN-inducible genes, including a key regulator of the IFNγ network, Irf1; 2) suppressed the expression of inducible nitric oxide synthase and type I IFN and 3) induced autophagy. These compounds may represent a basis for macrophage-directed therapies that fine-tune macrophage effector functions to combat intracellular pathogens and reduce inflammatory tissue damage. These therapies would be especially relevant to fighting drug-resistant pathogens, where improving host immunity may prove to be the ultimate resource.

No MeSH data available.


Related in: MedlinePlus