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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

Comparative activity and characterization of new rocaglates.(a) BMDM were treated with 1 μM compound (1-C9433, 2-C8809, 3-C10021, 4-C7564, 5-C7565, and 6-C10361) alone and in presence of 0.2 U/mL IFNγ for 24 hr. mRNA expression of Irf1, Irgm2, Irf5 and Ptgs2 was measured by qPCR. Data is presented relative to expression in untreated cells (set as 1). (b) BMDM were treated with 0.33 μM compounds (mentioned above) in presence and/or absence of 0.2 U/mL IFNγ for 24 hr. mRNA expression of Irf1, Irf5 and Ptgs2 was measured by qPCR. Data is presented relative to expression in 0.2U IFNγ treated cells for Irf1 and untreated for Irf5 and Ptgs2 (set as 1). (c) 293TR-FLuc cells were treated with 0.33 μM compounds (mentioned above) and luciferase activity was measured after 18 hrs. Data represents values from two independent experiments. (d) BMDM were treated with different doses of C9433 and C8809 in presence of 0.2 U/mL IFNγ for 24 hrs. mRNA expression of Irf1 was measured by qPCR. Data is presented relative to expression in 0.2U IFNγ treated cells (set as 1). (e) 293 T-FLuc cells were treated with different doses of C9433 and C8809 and the luciferase activity was measured after 18 hrs in triplicates. (f) BMDM were treated with C8809 and C9433 for 24 hrs in the presence and absence of 0.2 U/mL IFNγ and IRF1 induction was detected by immunoblotting. 20 U/mL IFNγ treated cells served as positive controls. (g) BMDM were treated with different doses of C8809 and C9433 for 6 hrs and probed with anti-LC3B mAb. A mix of chloroquine(10 μM) and rapamycin(500 nM) served as positive control (C). All immunoblots are representative of at least two independent experiments. (h) BMDM were treated with 15 ng/mL TNFα and 10 U/mL IFNγ for 24 hrs in the presence of different doses of C8809 and NO production was measured in triplicates. (i) BMDM were treated with 10 ng/ml TNFα in the presence of C8809 for 24 hrs. mRNA expression of IFNβ, IP10 and IL10 were measured by q-PCR. Data is calculated as % of gene expression relative to 10 ng/mL TNFα treated cells. All q-PCR results were normalized to expression of 18S and are representative of at least two independent experiments.
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f5: Comparative activity and characterization of new rocaglates.(a) BMDM were treated with 1 μM compound (1-C9433, 2-C8809, 3-C10021, 4-C7564, 5-C7565, and 6-C10361) alone and in presence of 0.2 U/mL IFNγ for 24 hr. mRNA expression of Irf1, Irgm2, Irf5 and Ptgs2 was measured by qPCR. Data is presented relative to expression in untreated cells (set as 1). (b) BMDM were treated with 0.33 μM compounds (mentioned above) in presence and/or absence of 0.2 U/mL IFNγ for 24 hr. mRNA expression of Irf1, Irf5 and Ptgs2 was measured by qPCR. Data is presented relative to expression in 0.2U IFNγ treated cells for Irf1 and untreated for Irf5 and Ptgs2 (set as 1). (c) 293TR-FLuc cells were treated with 0.33 μM compounds (mentioned above) and luciferase activity was measured after 18 hrs. Data represents values from two independent experiments. (d) BMDM were treated with different doses of C9433 and C8809 in presence of 0.2 U/mL IFNγ for 24 hrs. mRNA expression of Irf1 was measured by qPCR. Data is presented relative to expression in 0.2U IFNγ treated cells (set as 1). (e) 293 T-FLuc cells were treated with different doses of C9433 and C8809 and the luciferase activity was measured after 18 hrs in triplicates. (f) BMDM were treated with C8809 and C9433 for 24 hrs in the presence and absence of 0.2 U/mL IFNγ and IRF1 induction was detected by immunoblotting. 20 U/mL IFNγ treated cells served as positive controls. (g) BMDM were treated with different doses of C8809 and C9433 for 6 hrs and probed with anti-LC3B mAb. A mix of chloroquine(10 μM) and rapamycin(500 nM) served as positive control (C). All immunoblots are representative of at least two independent experiments. (h) BMDM were treated with 15 ng/mL TNFα and 10 U/mL IFNγ for 24 hrs in the presence of different doses of C8809 and NO production was measured in triplicates. (i) BMDM were treated with 10 ng/ml TNFα in the presence of C8809 for 24 hrs. mRNA expression of IFNβ, IP10 and IL10 were measured by q-PCR. Data is calculated as % of gene expression relative to 10 ng/mL TNFα treated cells. All q-PCR results were normalized to expression of 18S and are representative of at least two independent experiments.

Mentions: We screened an additional library of 69 rocaglate derivatives from the BU-CMD collection and identified 5 novel compounds, whose activity in primary macrophages was similar to C9433: at 1 μM they synergized with 0.2 U/mL of IFNγ as demonstrated by Irf1 and Irgm2 mRNA induction (Fig. 5a), and directly induced Irf5 and Ptgs2 mRNA expression (Fig. 5a). Next we ranked their synergistic activity at a lower concentration (0.3 μM): C8809 (#2) was the most active, while 10361 (#6) was the least active (Fig. 5b and Supplementary Fig. S5). However, the translation inhibition effect of C9433, C8809, and C10361 (rocaglaol, a derivative of rocaglamide A) at 0.3 μM was similar (Fig. 5c). Thus, the effect of rocaglates on gene expression did not completely parallel their inhibitory activity on protein translation.


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)

Comparative activity and characterization of new rocaglates.(a) BMDM were treated with 1 μM compound (1-C9433, 2-C8809, 3-C10021, 4-C7564, 5-C7565, and 6-C10361) alone and in presence of 0.2 U/mL IFNγ for 24 hr. mRNA expression of Irf1, Irgm2, Irf5 and Ptgs2 was measured by qPCR. Data is presented relative to expression in untreated cells (set as 1). (b) BMDM were treated with 0.33 μM compounds (mentioned above) in presence and/or absence of 0.2 U/mL IFNγ for 24 hr. mRNA expression of Irf1, Irf5 and Ptgs2 was measured by qPCR. Data is presented relative to expression in 0.2U IFNγ treated cells for Irf1 and untreated for Irf5 and Ptgs2 (set as 1). (c) 293TR-FLuc cells were treated with 0.33 μM compounds (mentioned above) and luciferase activity was measured after 18 hrs. Data represents values from two independent experiments. (d) BMDM were treated with different doses of C9433 and C8809 in presence of 0.2 U/mL IFNγ for 24 hrs. mRNA expression of Irf1 was measured by qPCR. Data is presented relative to expression in 0.2U IFNγ treated cells (set as 1). (e) 293 T-FLuc cells were treated with different doses of C9433 and C8809 and the luciferase activity was measured after 18 hrs in triplicates. (f) BMDM were treated with C8809 and C9433 for 24 hrs in the presence and absence of 0.2 U/mL IFNγ and IRF1 induction was detected by immunoblotting. 20 U/mL IFNγ treated cells served as positive controls. (g) BMDM were treated with different doses of C8809 and C9433 for 6 hrs and probed with anti-LC3B mAb. A mix of chloroquine(10 μM) and rapamycin(500 nM) served as positive control (C). All immunoblots are representative of at least two independent experiments. (h) BMDM were treated with 15 ng/mL TNFα and 10 U/mL IFNγ for 24 hrs in the presence of different doses of C8809 and NO production was measured in triplicates. (i) BMDM were treated with 10 ng/ml TNFα in the presence of C8809 for 24 hrs. mRNA expression of IFNβ, IP10 and IL10 were measured by q-PCR. Data is calculated as % of gene expression relative to 10 ng/mL TNFα treated cells. All q-PCR results were normalized to expression of 18S and are representative of at least two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4834551&req=5

f5: Comparative activity and characterization of new rocaglates.(a) BMDM were treated with 1 μM compound (1-C9433, 2-C8809, 3-C10021, 4-C7564, 5-C7565, and 6-C10361) alone and in presence of 0.2 U/mL IFNγ for 24 hr. mRNA expression of Irf1, Irgm2, Irf5 and Ptgs2 was measured by qPCR. Data is presented relative to expression in untreated cells (set as 1). (b) BMDM were treated with 0.33 μM compounds (mentioned above) in presence and/or absence of 0.2 U/mL IFNγ for 24 hr. mRNA expression of Irf1, Irf5 and Ptgs2 was measured by qPCR. Data is presented relative to expression in 0.2U IFNγ treated cells for Irf1 and untreated for Irf5 and Ptgs2 (set as 1). (c) 293TR-FLuc cells were treated with 0.33 μM compounds (mentioned above) and luciferase activity was measured after 18 hrs. Data represents values from two independent experiments. (d) BMDM were treated with different doses of C9433 and C8809 in presence of 0.2 U/mL IFNγ for 24 hrs. mRNA expression of Irf1 was measured by qPCR. Data is presented relative to expression in 0.2U IFNγ treated cells (set as 1). (e) 293 T-FLuc cells were treated with different doses of C9433 and C8809 and the luciferase activity was measured after 18 hrs in triplicates. (f) BMDM were treated with C8809 and C9433 for 24 hrs in the presence and absence of 0.2 U/mL IFNγ and IRF1 induction was detected by immunoblotting. 20 U/mL IFNγ treated cells served as positive controls. (g) BMDM were treated with different doses of C8809 and C9433 for 6 hrs and probed with anti-LC3B mAb. A mix of chloroquine(10 μM) and rapamycin(500 nM) served as positive control (C). All immunoblots are representative of at least two independent experiments. (h) BMDM were treated with 15 ng/mL TNFα and 10 U/mL IFNγ for 24 hrs in the presence of different doses of C8809 and NO production was measured in triplicates. (i) BMDM were treated with 10 ng/ml TNFα in the presence of C8809 for 24 hrs. mRNA expression of IFNβ, IP10 and IL10 were measured by q-PCR. Data is calculated as % of gene expression relative to 10 ng/mL TNFα treated cells. All q-PCR results were normalized to expression of 18S and are representative of at least two independent experiments.
Mentions: We screened an additional library of 69 rocaglate derivatives from the BU-CMD collection and identified 5 novel compounds, whose activity in primary macrophages was similar to C9433: at 1 μM they synergized with 0.2 U/mL of IFNγ as demonstrated by Irf1 and Irgm2 mRNA induction (Fig. 5a), and directly induced Irf5 and Ptgs2 mRNA expression (Fig. 5a). Next we ranked their synergistic activity at a lower concentration (0.3 μM): C8809 (#2) was the most active, while 10361 (#6) was the least active (Fig. 5b and Supplementary Fig. S5). However, the translation inhibition effect of C9433, C8809, and C10361 (rocaglaol, a derivative of rocaglamide A) at 0.3 μM was similar (Fig. 5c). Thus, the effect of rocaglates on gene expression did not completely parallel their inhibitory activity on protein translation.

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