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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

IFNγ regulates expression of Ipr1 at transcriptional and posttranscriptional levels in the nucleus of macrophages.(a) Expression of GFP-Ipr1 and endogenous Ipr1 in macrophage cell line J774A.1 clone J7-21. Nuclear extracts were prepared from J7-21 cells untreated (−) or treated with 1 μg/mL dox and/or 100 U/mL IFNγ for 24 hrs. GFP-Ipr1 and endogenous Ipr1 proteins were detected by immunoblotting; (b) Nuclear extracts from J7-21 cells treated with 1 μg/mL dox and/or 100 U/mL IFNγ for indicated times were prepared and GFP-Ipr1 expression was detected by immunoblotting. (c) Nuclei of J7-21 cells treated with 1 μg/mL dox and 100 U/mL IFNγ for 24 hrs were fractionated into nucleoplasmic and chromatin fractions and endogenous Ipr1 and GFP-Ipr1 was detected by immunoblotting. All immunoblots were carried out using Ipr1 specific rabbit polyclonal antibodies. (d) Immunofluorescence of J7-21 cells treated with 1 μg/mL dox alone and in presence of 100 U/mL IFNγ for 24 hrs for GFP-Ipr1 detection (FITC channel). (e) J7-21 cells were treated with 1 μg/mL dox and 100 U/mL IFNγ for 24 hrs and stained with Ipr1-specific monoclonal antibody (red, central panel), eGFP-Ipr1 is green (left panel) and merged image is yellow (right panel). (f) Real time RT-PCR analysis of the kinetics of Ipr1 mRNA expression in primary macrophages (C57BL/6 J BMDMs) after treatment with10 U/mL IFNγ for indicated times. (g) Dose dependent effect of IFNγ on Ipr1 mRNA expression in primary macrophages. B6 BMDMs were treated with indicated doses of IFNγ for 18 hrs. Ipr1 mRNA expression was determined using real-time RT-PCR, normalized to expression of RPS17 mRNA and presented relative to expression in untreated cells (set as 1). All qPCR results represent data from two independent experiments. (h) Top panel - Ipr1 protein expression in primary macrophages. Immunoblot analysis of nuclear and cytoplasmic extracts of C57BL/6 BMDM treated with 10 U/mL of IFNγ and 100 U/mL IFNβ for 24 hrs using Ipr1 specific polyclonal antibodies. Immunoblots represent data from at least two independent experiments. Lower panel - Immunofluorescence of B6 BMDMs stimulated with 10 U/mL IFNγ for 24 hrs showing nuclear localization of Ipr1. Cells were stained with anti-Ipr1 monoclonal antibody (red); nuclei are counterstained with DAPI (blue). All microscopic images represent data from at least two independent experiments.
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f1: IFNγ regulates expression of Ipr1 at transcriptional and posttranscriptional levels in the nucleus of macrophages.(a) Expression of GFP-Ipr1 and endogenous Ipr1 in macrophage cell line J774A.1 clone J7-21. Nuclear extracts were prepared from J7-21 cells untreated (−) or treated with 1 μg/mL dox and/or 100 U/mL IFNγ for 24 hrs. GFP-Ipr1 and endogenous Ipr1 proteins were detected by immunoblotting; (b) Nuclear extracts from J7-21 cells treated with 1 μg/mL dox and/or 100 U/mL IFNγ for indicated times were prepared and GFP-Ipr1 expression was detected by immunoblotting. (c) Nuclei of J7-21 cells treated with 1 μg/mL dox and 100 U/mL IFNγ for 24 hrs were fractionated into nucleoplasmic and chromatin fractions and endogenous Ipr1 and GFP-Ipr1 was detected by immunoblotting. All immunoblots were carried out using Ipr1 specific rabbit polyclonal antibodies. (d) Immunofluorescence of J7-21 cells treated with 1 μg/mL dox alone and in presence of 100 U/mL IFNγ for 24 hrs for GFP-Ipr1 detection (FITC channel). (e) J7-21 cells were treated with 1 μg/mL dox and 100 U/mL IFNγ for 24 hrs and stained with Ipr1-specific monoclonal antibody (red, central panel), eGFP-Ipr1 is green (left panel) and merged image is yellow (right panel). (f) Real time RT-PCR analysis of the kinetics of Ipr1 mRNA expression in primary macrophages (C57BL/6 J BMDMs) after treatment with10 U/mL IFNγ for indicated times. (g) Dose dependent effect of IFNγ on Ipr1 mRNA expression in primary macrophages. B6 BMDMs were treated with indicated doses of IFNγ for 18 hrs. Ipr1 mRNA expression was determined using real-time RT-PCR, normalized to expression of RPS17 mRNA and presented relative to expression in untreated cells (set as 1). All qPCR results represent data from two independent experiments. (h) Top panel - Ipr1 protein expression in primary macrophages. Immunoblot analysis of nuclear and cytoplasmic extracts of C57BL/6 BMDM treated with 10 U/mL of IFNγ and 100 U/mL IFNβ for 24 hrs using Ipr1 specific polyclonal antibodies. Immunoblots represent data from at least two independent experiments. Lower panel - Immunofluorescence of B6 BMDMs stimulated with 10 U/mL IFNγ for 24 hrs showing nuclear localization of Ipr1. Cells were stained with anti-Ipr1 monoclonal antibody (red); nuclei are counterstained with DAPI (blue). All microscopic images represent data from at least two independent experiments.

Mentions: For inducible expression of GFP-tagged Ipr1 fusion protein in the mouse macrophage cell line J774.A1, we used a doxycycline-inducible promoter and lentiviral delivery system as described elsewhere21. Clones that displayed no detectable basal GFP and high levels of inducible GFP-Ipr1 expression, were identified using flow cytometry. One of those clones (J7-21) was used for subsequent analyses and assay development. The inducible GFP-Ipr1 expression was confirmed using Ipr1-specific rabbit polyclonal (Fig. 1a and S1) and mouse monoclonal antibodies developed in our laboratory (Fig. 1e,h).


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)

IFNγ regulates expression of Ipr1 at transcriptional and posttranscriptional levels in the nucleus of macrophages.(a) Expression of GFP-Ipr1 and endogenous Ipr1 in macrophage cell line J774A.1 clone J7-21. Nuclear extracts were prepared from J7-21 cells untreated (−) or treated with 1 μg/mL dox and/or 100 U/mL IFNγ for 24 hrs. GFP-Ipr1 and endogenous Ipr1 proteins were detected by immunoblotting; (b) Nuclear extracts from J7-21 cells treated with 1 μg/mL dox and/or 100 U/mL IFNγ for indicated times were prepared and GFP-Ipr1 expression was detected by immunoblotting. (c) Nuclei of J7-21 cells treated with 1 μg/mL dox and 100 U/mL IFNγ for 24 hrs were fractionated into nucleoplasmic and chromatin fractions and endogenous Ipr1 and GFP-Ipr1 was detected by immunoblotting. All immunoblots were carried out using Ipr1 specific rabbit polyclonal antibodies. (d) Immunofluorescence of J7-21 cells treated with 1 μg/mL dox alone and in presence of 100 U/mL IFNγ for 24 hrs for GFP-Ipr1 detection (FITC channel). (e) J7-21 cells were treated with 1 μg/mL dox and 100 U/mL IFNγ for 24 hrs and stained with Ipr1-specific monoclonal antibody (red, central panel), eGFP-Ipr1 is green (left panel) and merged image is yellow (right panel). (f) Real time RT-PCR analysis of the kinetics of Ipr1 mRNA expression in primary macrophages (C57BL/6 J BMDMs) after treatment with10 U/mL IFNγ for indicated times. (g) Dose dependent effect of IFNγ on Ipr1 mRNA expression in primary macrophages. B6 BMDMs were treated with indicated doses of IFNγ for 18 hrs. Ipr1 mRNA expression was determined using real-time RT-PCR, normalized to expression of RPS17 mRNA and presented relative to expression in untreated cells (set as 1). All qPCR results represent data from two independent experiments. (h) Top panel - Ipr1 protein expression in primary macrophages. Immunoblot analysis of nuclear and cytoplasmic extracts of C57BL/6 BMDM treated with 10 U/mL of IFNγ and 100 U/mL IFNβ for 24 hrs using Ipr1 specific polyclonal antibodies. Immunoblots represent data from at least two independent experiments. Lower panel - Immunofluorescence of B6 BMDMs stimulated with 10 U/mL IFNγ for 24 hrs showing nuclear localization of Ipr1. Cells were stained with anti-Ipr1 monoclonal antibody (red); nuclei are counterstained with DAPI (blue). All microscopic images represent data from at least two independent experiments.
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f1: IFNγ regulates expression of Ipr1 at transcriptional and posttranscriptional levels in the nucleus of macrophages.(a) Expression of GFP-Ipr1 and endogenous Ipr1 in macrophage cell line J774A.1 clone J7-21. Nuclear extracts were prepared from J7-21 cells untreated (−) or treated with 1 μg/mL dox and/or 100 U/mL IFNγ for 24 hrs. GFP-Ipr1 and endogenous Ipr1 proteins were detected by immunoblotting; (b) Nuclear extracts from J7-21 cells treated with 1 μg/mL dox and/or 100 U/mL IFNγ for indicated times were prepared and GFP-Ipr1 expression was detected by immunoblotting. (c) Nuclei of J7-21 cells treated with 1 μg/mL dox and 100 U/mL IFNγ for 24 hrs were fractionated into nucleoplasmic and chromatin fractions and endogenous Ipr1 and GFP-Ipr1 was detected by immunoblotting. All immunoblots were carried out using Ipr1 specific rabbit polyclonal antibodies. (d) Immunofluorescence of J7-21 cells treated with 1 μg/mL dox alone and in presence of 100 U/mL IFNγ for 24 hrs for GFP-Ipr1 detection (FITC channel). (e) J7-21 cells were treated with 1 μg/mL dox and 100 U/mL IFNγ for 24 hrs and stained with Ipr1-specific monoclonal antibody (red, central panel), eGFP-Ipr1 is green (left panel) and merged image is yellow (right panel). (f) Real time RT-PCR analysis of the kinetics of Ipr1 mRNA expression in primary macrophages (C57BL/6 J BMDMs) after treatment with10 U/mL IFNγ for indicated times. (g) Dose dependent effect of IFNγ on Ipr1 mRNA expression in primary macrophages. B6 BMDMs were treated with indicated doses of IFNγ for 18 hrs. Ipr1 mRNA expression was determined using real-time RT-PCR, normalized to expression of RPS17 mRNA and presented relative to expression in untreated cells (set as 1). All qPCR results represent data from two independent experiments. (h) Top panel - Ipr1 protein expression in primary macrophages. Immunoblot analysis of nuclear and cytoplasmic extracts of C57BL/6 BMDM treated with 10 U/mL of IFNγ and 100 U/mL IFNβ for 24 hrs using Ipr1 specific polyclonal antibodies. Immunoblots represent data from at least two independent experiments. Lower panel - Immunofluorescence of B6 BMDMs stimulated with 10 U/mL IFNγ for 24 hrs showing nuclear localization of Ipr1. Cells were stained with anti-Ipr1 monoclonal antibody (red); nuclei are counterstained with DAPI (blue). All microscopic images represent data from at least two independent experiments.
Mentions: For inducible expression of GFP-tagged Ipr1 fusion protein in the mouse macrophage cell line J774.A1, we used a doxycycline-inducible promoter and lentiviral delivery system as described elsewhere21. Clones that displayed no detectable basal GFP and high levels of inducible GFP-Ipr1 expression, were identified using flow cytometry. One of those clones (J7-21) was used for subsequent analyses and assay development. The inducible GFP-Ipr1 expression was confirmed using Ipr1-specific rabbit polyclonal (Fig. 1a and S1) and mouse monoclonal antibodies developed in our laboratory (Fig. 1e,h).

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