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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

Comaparative activity of known rocaglates and translational inhibitors.(a) BMDM were treated with 0.33 μM compounds in presence of 0.2 U/mL IFNγ for 24 hr. mRNA expression of Irf1 was measured by qPCR and normalized to expression of 18 S. Data is presented relative to expression in 0.2U IFNγ treated cells (set as 1) and represents results from two independent experiments. (b) BMDM were treated with silvestrol and RHT at concentrations shown for 24 hrs and % of PI positive cells were calculated. Data is represented as % of survival of two independent experiments performed in duplicates. (c) BMDM were treated with 15 ng/mL TNFα and 10 U/mL IFNγ for 24 hrs in the presence and absence of 1 μM compounds and production of NO (assayed as NO2−) was determined. All measurements for NO production were performed in triplicates. (d) BMDM were treated with 1 μM compounds for 6 hrs and autophagy was determined by increase of LC3B-II to LC3B-I ratio by immunoblotting. Blots represent data of two independent experiments. 293 T-FLuc cells were treated with different doses of (e) silvestrol, RHT, exo-RHT and ent-RHT and (f) cycloheximide and rapamycin. The luciferase activity was measured after 18 hrs. Data represents values from experiment performed in triplicates. (g) BMDM were treated with cycloheximide and rapamycin at concentrations shown for 24 hrs and % of PI positive cells were calculated. Data is represented as % of survival of two independent experiments performed in duplicates. (h) BMDM were treated with 0.33 μM compounds in presence of 0.2 U/mL IFNγ for 24 hr. mRNA expression of Irf1, Irf5 and Ptgs2 was measured by qPCR and normalized to expression of 18 S. Data is presented relative to expression in 0.2U IFNγ treated cells (set as 1) and represents results from two independent experiments. (i) BMDM were treated with 0.33, 1 and 3.3 μM rapamycin in presence of 0.2 U/mL IFNγ for 24 hr. mRNA expression of Irf1, Irf5 and Ptgs2 was measured by qPCR and normalized to expression of 18S. Data is presented relative to expression in 0.2U IFNγ treated cells (set as 1) and represents results from two independent experiments.
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f7: Comaparative activity of known rocaglates and translational inhibitors.(a) BMDM were treated with 0.33 μM compounds in presence of 0.2 U/mL IFNγ for 24 hr. mRNA expression of Irf1 was measured by qPCR and normalized to expression of 18 S. Data is presented relative to expression in 0.2U IFNγ treated cells (set as 1) and represents results from two independent experiments. (b) BMDM were treated with silvestrol and RHT at concentrations shown for 24 hrs and % of PI positive cells were calculated. Data is represented as % of survival of two independent experiments performed in duplicates. (c) BMDM were treated with 15 ng/mL TNFα and 10 U/mL IFNγ for 24 hrs in the presence and absence of 1 μM compounds and production of NO (assayed as NO2−) was determined. All measurements for NO production were performed in triplicates. (d) BMDM were treated with 1 μM compounds for 6 hrs and autophagy was determined by increase of LC3B-II to LC3B-I ratio by immunoblotting. Blots represent data of two independent experiments. 293 T-FLuc cells were treated with different doses of (e) silvestrol, RHT, exo-RHT and ent-RHT and (f) cycloheximide and rapamycin. The luciferase activity was measured after 18 hrs. Data represents values from experiment performed in triplicates. (g) BMDM were treated with cycloheximide and rapamycin at concentrations shown for 24 hrs and % of PI positive cells were calculated. Data is represented as % of survival of two independent experiments performed in duplicates. (h) BMDM were treated with 0.33 μM compounds in presence of 0.2 U/mL IFNγ for 24 hr. mRNA expression of Irf1, Irf5 and Ptgs2 was measured by qPCR and normalized to expression of 18 S. Data is presented relative to expression in 0.2U IFNγ treated cells (set as 1) and represents results from two independent experiments. (i) BMDM were treated with 0.33, 1 and 3.3 μM rapamycin in presence of 0.2 U/mL IFNγ for 24 hr. mRNA expression of Irf1, Irf5 and Ptgs2 was measured by qPCR and normalized to expression of 18S. Data is presented relative to expression in 0.2U IFNγ treated cells (set as 1) and represents results from two independent experiments.

Mentions: In order to determine whether translation inhibition was necessary, we tested silvestrol and rohitinib (RHT), two well-studied rocaglates possessing both potent translation inhibition and cytotoxic activities. Both are known to suppress the cap-dependent translation initiation complex eIF4F by binding to and inhibiting an RNA helicase (eIF4A)224243. Both RHT and silvestrol showed significant toxicity at the highest two doses (1 and 3.3 μM) in primary macrophages (Fig. 7b) and potent translation inhibition (EC50 for silvestrol is 40 nM, for RHT it is 10 nM, Fig. 7e). Notably silvestrol was more toxic to primary macrophages, but was found to be less active in comparison to RHT. At 0.33 μM, the highest concentration that was not cytotoxic for primary macrophages within 24 hrs, both compounds synergized with IFNγ to induce Irf1 mRNA expression (Fig. 7a).


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)

Comaparative activity of known rocaglates and translational inhibitors.(a) BMDM were treated with 0.33 μM compounds in presence of 0.2 U/mL IFNγ for 24 hr. mRNA expression of Irf1 was measured by qPCR and normalized to expression of 18 S. Data is presented relative to expression in 0.2U IFNγ treated cells (set as 1) and represents results from two independent experiments. (b) BMDM were treated with silvestrol and RHT at concentrations shown for 24 hrs and % of PI positive cells were calculated. Data is represented as % of survival of two independent experiments performed in duplicates. (c) BMDM were treated with 15 ng/mL TNFα and 10 U/mL IFNγ for 24 hrs in the presence and absence of 1 μM compounds and production of NO (assayed as NO2−) was determined. All measurements for NO production were performed in triplicates. (d) BMDM were treated with 1 μM compounds for 6 hrs and autophagy was determined by increase of LC3B-II to LC3B-I ratio by immunoblotting. Blots represent data of two independent experiments. 293 T-FLuc cells were treated with different doses of (e) silvestrol, RHT, exo-RHT and ent-RHT and (f) cycloheximide and rapamycin. The luciferase activity was measured after 18 hrs. Data represents values from experiment performed in triplicates. (g) BMDM were treated with cycloheximide and rapamycin at concentrations shown for 24 hrs and % of PI positive cells were calculated. Data is represented as % of survival of two independent experiments performed in duplicates. (h) BMDM were treated with 0.33 μM compounds in presence of 0.2 U/mL IFNγ for 24 hr. mRNA expression of Irf1, Irf5 and Ptgs2 was measured by qPCR and normalized to expression of 18 S. Data is presented relative to expression in 0.2U IFNγ treated cells (set as 1) and represents results from two independent experiments. (i) BMDM were treated with 0.33, 1 and 3.3 μM rapamycin in presence of 0.2 U/mL IFNγ for 24 hr. mRNA expression of Irf1, Irf5 and Ptgs2 was measured by qPCR and normalized to expression of 18S. Data is presented relative to expression in 0.2U IFNγ treated cells (set as 1) and represents results from two independent experiments.
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Related In: Results  -  Collection

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f7: Comaparative activity of known rocaglates and translational inhibitors.(a) BMDM were treated with 0.33 μM compounds in presence of 0.2 U/mL IFNγ for 24 hr. mRNA expression of Irf1 was measured by qPCR and normalized to expression of 18 S. Data is presented relative to expression in 0.2U IFNγ treated cells (set as 1) and represents results from two independent experiments. (b) BMDM were treated with silvestrol and RHT at concentrations shown for 24 hrs and % of PI positive cells were calculated. Data is represented as % of survival of two independent experiments performed in duplicates. (c) BMDM were treated with 15 ng/mL TNFα and 10 U/mL IFNγ for 24 hrs in the presence and absence of 1 μM compounds and production of NO (assayed as NO2−) was determined. All measurements for NO production were performed in triplicates. (d) BMDM were treated with 1 μM compounds for 6 hrs and autophagy was determined by increase of LC3B-II to LC3B-I ratio by immunoblotting. Blots represent data of two independent experiments. 293 T-FLuc cells were treated with different doses of (e) silvestrol, RHT, exo-RHT and ent-RHT and (f) cycloheximide and rapamycin. The luciferase activity was measured after 18 hrs. Data represents values from experiment performed in triplicates. (g) BMDM were treated with cycloheximide and rapamycin at concentrations shown for 24 hrs and % of PI positive cells were calculated. Data is represented as % of survival of two independent experiments performed in duplicates. (h) BMDM were treated with 0.33 μM compounds in presence of 0.2 U/mL IFNγ for 24 hr. mRNA expression of Irf1, Irf5 and Ptgs2 was measured by qPCR and normalized to expression of 18 S. Data is presented relative to expression in 0.2U IFNγ treated cells (set as 1) and represents results from two independent experiments. (i) BMDM were treated with 0.33, 1 and 3.3 μM rapamycin in presence of 0.2 U/mL IFNγ for 24 hr. mRNA expression of Irf1, Irf5 and Ptgs2 was measured by qPCR and normalized to expression of 18S. Data is presented relative to expression in 0.2U IFNγ treated cells (set as 1) and represents results from two independent experiments.
Mentions: In order to determine whether translation inhibition was necessary, we tested silvestrol and rohitinib (RHT), two well-studied rocaglates possessing both potent translation inhibition and cytotoxic activities. Both are known to suppress the cap-dependent translation initiation complex eIF4F by binding to and inhibiting an RNA helicase (eIF4A)224243. Both RHT and silvestrol showed significant toxicity at the highest two doses (1 and 3.3 μM) in primary macrophages (Fig. 7b) and potent translation inhibition (EC50 for silvestrol is 40 nM, for RHT it is 10 nM, Fig. 7e). Notably silvestrol was more toxic to primary macrophages, but was found to be less active in comparison to RHT. At 0.33 μM, the highest concentration that was not cytotoxic for primary macrophages within 24 hrs, both compounds synergized with IFNγ to induce Irf1 mRNA expression (Fig. 7a).

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