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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

Development and validation of GFP-Ipr1 based screen for small molecules.(a) J7-21 cells were treated with 0.2 U and 10 U/mL IFNγ in presence of 1 μg/mL dox for 24 hrs and GFP-Ipr1 expression measured. The gating strategy (mentioned in text) was used to quantify GFP-Ipr1 expression. (b) J7-21 cells were treated with 1 μg/mL dox alone (0 U/mL IFNγ) or in presence of 100 U/mL IFNγ for indicated times and fluorescence intensity of GFP-Ipr1 was measured. (c) J7-21 cells were primed with 1 μg/mL dox and 100 U/mL IFNγ for 24 hrs and then subjected to the following treatments for additional 72 hrs: 1.dox and IFNγ kept throughout, 2.dox kept and IFNγ removed, 3.dox removed and IFNγ kept, 4.dox and IFNγ removed. GFP-Ipr1 expression was measured using the celigo cytometer. (d) J7-21 cells were primed with 5, 25 and 100 U/mL IFNγ for 24 hrs and then removed. 1 μg/mL dox was added and expression of GFP-Ipr1 was measured after 24, 48 and 72 hrs. (e) J7-21 cells were treated with different doses of IFNγ for 24 hr. The next day dox(1 μg/mL) was added for 24 hrs and % of cells expressing GFP-Ipr1 was calculated. A schematic representation of the experiment for C, D and E are shown. (f) J7-21 cells were treated with different doses of AM580 and PMA in presence of IFNγ(0.2 U/mL) and 1 μg/mL dox and fluorescent intensity of GFP-Ipr1 expression was measured. Cells expressing GFP-Ipr1 was calculated as % expression with respect to 10 U/mL IFNγ. All graphs are representative of at least two independent experiments. (g) Immunoblot analysis of whole-cell extracts of J7-21 cells treated with 8 μM PMA and 10 μM AM580 in presence and absence of 0.2 U/mL IFNγ and 1 μg/mL dox for 24 hrs. Blots were probed with Ipr1 polyclonal antibodies. Blots are representative of two independent experiments. Fold induction of GFP-Ipr1 was calculated relative to expression in untreated cells (set as 1) by densitometric analysis after normalizing it to loading control β-actin. (h) Microscopy of BMDM from C57BL/6 mice cultured with 2 μM PMA and 3.3 μM AM580 in presence of 0.2 U/mL IFNγ for 24 hrs and stained with Ipr1 monoclonal antibody(red). Images represent data from two independent experiments performed in duplicates.
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f2: Development and validation of GFP-Ipr1 based screen for small molecules.(a) J7-21 cells were treated with 0.2 U and 10 U/mL IFNγ in presence of 1 μg/mL dox for 24 hrs and GFP-Ipr1 expression measured. The gating strategy (mentioned in text) was used to quantify GFP-Ipr1 expression. (b) J7-21 cells were treated with 1 μg/mL dox alone (0 U/mL IFNγ) or in presence of 100 U/mL IFNγ for indicated times and fluorescence intensity of GFP-Ipr1 was measured. (c) J7-21 cells were primed with 1 μg/mL dox and 100 U/mL IFNγ for 24 hrs and then subjected to the following treatments for additional 72 hrs: 1.dox and IFNγ kept throughout, 2.dox kept and IFNγ removed, 3.dox removed and IFNγ kept, 4.dox and IFNγ removed. GFP-Ipr1 expression was measured using the celigo cytometer. (d) J7-21 cells were primed with 5, 25 and 100 U/mL IFNγ for 24 hrs and then removed. 1 μg/mL dox was added and expression of GFP-Ipr1 was measured after 24, 48 and 72 hrs. (e) J7-21 cells were treated with different doses of IFNγ for 24 hr. The next day dox(1 μg/mL) was added for 24 hrs and % of cells expressing GFP-Ipr1 was calculated. A schematic representation of the experiment for C, D and E are shown. (f) J7-21 cells were treated with different doses of AM580 and PMA in presence of IFNγ(0.2 U/mL) and 1 μg/mL dox and fluorescent intensity of GFP-Ipr1 expression was measured. Cells expressing GFP-Ipr1 was calculated as % expression with respect to 10 U/mL IFNγ. All graphs are representative of at least two independent experiments. (g) Immunoblot analysis of whole-cell extracts of J7-21 cells treated with 8 μM PMA and 10 μM AM580 in presence and absence of 0.2 U/mL IFNγ and 1 μg/mL dox for 24 hrs. Blots were probed with Ipr1 polyclonal antibodies. Blots are representative of two independent experiments. Fold induction of GFP-Ipr1 was calculated relative to expression in untreated cells (set as 1) by densitometric analysis after normalizing it to loading control β-actin. (h) Microscopy of BMDM from C57BL/6 mice cultured with 2 μM PMA and 3.3 μM AM580 in presence of 0.2 U/mL IFNγ for 24 hrs and stained with Ipr1 monoclonal antibody(red). Images represent data from two independent experiments performed in duplicates.

Mentions: To develop the screening protocol, we performed time course and dose-response analyses of GFP-Ipr1 expression using an automated cytometry system (Celigo) in a 96-well format. After treatment with IFNγ and Dox, J7-21 cells were fixed, stained with DAPI and analyzed using Celigo on GFP and DAPI channels. GFP fluorescence within nuclear areas delineated by DAPI staining was plotted against total the DAPI signal as presented in Fig. 2a. Gating on the upper left quadrant that contains diploid cells with elevated expression of GFP-Ipr1 in the nuclei was used to normalize the GFP signal against DNA content and provided more specific measure of GFP-Ipr1 induction as compared to total GFP signal. We used this gating strategy to determine thresholds for enumeration of GFP-positive nuclei in subsequent experiments and high throughoutput screening.


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)

Development and validation of GFP-Ipr1 based screen for small molecules.(a) J7-21 cells were treated with 0.2 U and 10 U/mL IFNγ in presence of 1 μg/mL dox for 24 hrs and GFP-Ipr1 expression measured. The gating strategy (mentioned in text) was used to quantify GFP-Ipr1 expression. (b) J7-21 cells were treated with 1 μg/mL dox alone (0 U/mL IFNγ) or in presence of 100 U/mL IFNγ for indicated times and fluorescence intensity of GFP-Ipr1 was measured. (c) J7-21 cells were primed with 1 μg/mL dox and 100 U/mL IFNγ for 24 hrs and then subjected to the following treatments for additional 72 hrs: 1.dox and IFNγ kept throughout, 2.dox kept and IFNγ removed, 3.dox removed and IFNγ kept, 4.dox and IFNγ removed. GFP-Ipr1 expression was measured using the celigo cytometer. (d) J7-21 cells were primed with 5, 25 and 100 U/mL IFNγ for 24 hrs and then removed. 1 μg/mL dox was added and expression of GFP-Ipr1 was measured after 24, 48 and 72 hrs. (e) J7-21 cells were treated with different doses of IFNγ for 24 hr. The next day dox(1 μg/mL) was added for 24 hrs and % of cells expressing GFP-Ipr1 was calculated. A schematic representation of the experiment for C, D and E are shown. (f) J7-21 cells were treated with different doses of AM580 and PMA in presence of IFNγ(0.2 U/mL) and 1 μg/mL dox and fluorescent intensity of GFP-Ipr1 expression was measured. Cells expressing GFP-Ipr1 was calculated as % expression with respect to 10 U/mL IFNγ. All graphs are representative of at least two independent experiments. (g) Immunoblot analysis of whole-cell extracts of J7-21 cells treated with 8 μM PMA and 10 μM AM580 in presence and absence of 0.2 U/mL IFNγ and 1 μg/mL dox for 24 hrs. Blots were probed with Ipr1 polyclonal antibodies. Blots are representative of two independent experiments. Fold induction of GFP-Ipr1 was calculated relative to expression in untreated cells (set as 1) by densitometric analysis after normalizing it to loading control β-actin. (h) Microscopy of BMDM from C57BL/6 mice cultured with 2 μM PMA and 3.3 μM AM580 in presence of 0.2 U/mL IFNγ for 24 hrs and stained with Ipr1 monoclonal antibody(red). Images represent data from two independent experiments performed in duplicates.
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Related In: Results  -  Collection

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Show All Figures
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f2: Development and validation of GFP-Ipr1 based screen for small molecules.(a) J7-21 cells were treated with 0.2 U and 10 U/mL IFNγ in presence of 1 μg/mL dox for 24 hrs and GFP-Ipr1 expression measured. The gating strategy (mentioned in text) was used to quantify GFP-Ipr1 expression. (b) J7-21 cells were treated with 1 μg/mL dox alone (0 U/mL IFNγ) or in presence of 100 U/mL IFNγ for indicated times and fluorescence intensity of GFP-Ipr1 was measured. (c) J7-21 cells were primed with 1 μg/mL dox and 100 U/mL IFNγ for 24 hrs and then subjected to the following treatments for additional 72 hrs: 1.dox and IFNγ kept throughout, 2.dox kept and IFNγ removed, 3.dox removed and IFNγ kept, 4.dox and IFNγ removed. GFP-Ipr1 expression was measured using the celigo cytometer. (d) J7-21 cells were primed with 5, 25 and 100 U/mL IFNγ for 24 hrs and then removed. 1 μg/mL dox was added and expression of GFP-Ipr1 was measured after 24, 48 and 72 hrs. (e) J7-21 cells were treated with different doses of IFNγ for 24 hr. The next day dox(1 μg/mL) was added for 24 hrs and % of cells expressing GFP-Ipr1 was calculated. A schematic representation of the experiment for C, D and E are shown. (f) J7-21 cells were treated with different doses of AM580 and PMA in presence of IFNγ(0.2 U/mL) and 1 μg/mL dox and fluorescent intensity of GFP-Ipr1 expression was measured. Cells expressing GFP-Ipr1 was calculated as % expression with respect to 10 U/mL IFNγ. All graphs are representative of at least two independent experiments. (g) Immunoblot analysis of whole-cell extracts of J7-21 cells treated with 8 μM PMA and 10 μM AM580 in presence and absence of 0.2 U/mL IFNγ and 1 μg/mL dox for 24 hrs. Blots were probed with Ipr1 polyclonal antibodies. Blots are representative of two independent experiments. Fold induction of GFP-Ipr1 was calculated relative to expression in untreated cells (set as 1) by densitometric analysis after normalizing it to loading control β-actin. (h) Microscopy of BMDM from C57BL/6 mice cultured with 2 μM PMA and 3.3 μM AM580 in presence of 0.2 U/mL IFNγ for 24 hrs and stained with Ipr1 monoclonal antibody(red). Images represent data from two independent experiments performed in duplicates.
Mentions: To develop the screening protocol, we performed time course and dose-response analyses of GFP-Ipr1 expression using an automated cytometry system (Celigo) in a 96-well format. After treatment with IFNγ and Dox, J7-21 cells were fixed, stained with DAPI and analyzed using Celigo on GFP and DAPI channels. GFP fluorescence within nuclear areas delineated by DAPI staining was plotted against total the DAPI signal as presented in Fig. 2a. Gating on the upper left quadrant that contains diploid cells with elevated expression of GFP-Ipr1 in the nuclei was used to normalize the GFP signal against DNA content and provided more specific measure of GFP-Ipr1 induction as compared to total GFP signal. We used this gating strategy to determine thresholds for enumeration of GFP-positive nuclei in subsequent experiments and high throughoutput screening.

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