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IRF8 directs stress-induced autophagy in macrophages and promotes clearance of Listeria monocytogenes.

Gupta M, Shin DM, Ramakrishna L, Goussetis DJ, Platanias LC, Xiong H, Morse HC, Ozato K - Nat Commun (2015)

Bottom Line: Autophagy, activated by many stresses, plays a critical role in innate immune responses.Here we show that interferon regulatory factor 8 (IRF8) is required for the expression of autophagy-related genes in dendritic cells.IRF8 directly activates many genes involved in various steps of autophagy, promoting autophagosome formation and lysosomal fusion.

View Article: PubMed Central - PubMed

Affiliation: Program in Genomics of Differentiation, NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
Autophagy, activated by many stresses, plays a critical role in innate immune responses. Here we show that interferon regulatory factor 8 (IRF8) is required for the expression of autophagy-related genes in dendritic cells. Furthermore in macrophages, IRF8 is induced by multiple autophagy-inducing stresses, including IFNγ and Toll-like receptor stimulation, bacterial infection, starvation and by macrophage colony-stimulating factor. IRF8 directly activates many genes involved in various steps of autophagy, promoting autophagosome formation and lysosomal fusion. Consequently, Irf8(-/-) macrophages are deficient in autophagic activity, and excessively accumulate SQSTM1 and ubiquitin-bound proteins. We show that clearance of Listeria monocytogenes in macrophages requires IRF8-dependent activation of autophagy genes and subsequent autophagic capturing and degradation of Listeria antigens. These processes are defective in Irf8(-/-) macrophages where uninhibited bacterial growth ensues. Together these data suggest that IRF8 is a major autophagy regulator in macrophages, essential for macrophage maturation, survival and innate immune responses.

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Defective autophagosome formation in Irf8-/- MΦs(a) LC3 vesicles were visualized in WT and Irf8-/- MΦs expressing mCherry-EGFP-LC3 vector, without (UT) or with IFNγ/TLR stimulation for 8 h. Bafilomycin A1 (200 nM) was added for final 2 h. Cells were counterstained for DNA (blue). The scale bar: 20 μm. Below: The number of cells with more than five mCherry-positive vesicles was counted by microscopic inspection of more than 200 cells. The values represent the percentage of cells with fluorescent vesicles. **p-value ≤0.01 (Student's t-test). See Supplementary Fig. 3a for endogenous staining of LC3.(b) WT and Irf8-/- MΦs treated with IFNγ overnight followed by TLR ligands for 8 h was inspected by transmission electron microscopy. The bracketed region in the left panel was enlarged in the right panel. Arrows indicate autophagic vacuoles. The scale bar: 0.5 μm.(c) Reduced LC3I to LC3II conversion in Irf8-/- MΦs. WT and Irf8-/- MΦs were treated with IFNγ/TLR as above with bafilomycin A1 (200 nM) treatment for the final 2 h. Immunoblot analysis was performed with 10 μg of extracts with β-Tubulin as a control. Right panel: The amounts of LC3II in three independent samples were quantified using the ImageJ software. *p-value ≤0.05 and **p-value ≤0.01(Student's t-test). See Supplementary Fig. 3b for LC3 amount in the absence of bafilomycin A1.(d) Membrane bound LC3 in WT and Irf8-/- MΦs treated with IFNγ/TLR was detected by flow cytometry. Bafilomycin A1 (200 nM) was added for the final 2 h and. The histogram is a representative of three independent experiments. BA1: Bafilomycin A1. See Supplementary Fig. 3c for LC3 amount in the absence of bafilomycin A1.(e) Immunoblot detection of ATG5-ATG12 conjugate. WT and Irf8-/- MΦs were treated as above and immunoblot detection of ATG5-ATG12 conjugate proteins was performed. Ten microgram of extracts was tested with antibody against ATG5 or β-Tubulin.
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Figure 3: Defective autophagosome formation in Irf8-/- MΦs(a) LC3 vesicles were visualized in WT and Irf8-/- MΦs expressing mCherry-EGFP-LC3 vector, without (UT) or with IFNγ/TLR stimulation for 8 h. Bafilomycin A1 (200 nM) was added for final 2 h. Cells were counterstained for DNA (blue). The scale bar: 20 μm. Below: The number of cells with more than five mCherry-positive vesicles was counted by microscopic inspection of more than 200 cells. The values represent the percentage of cells with fluorescent vesicles. **p-value ≤0.01 (Student's t-test). See Supplementary Fig. 3a for endogenous staining of LC3.(b) WT and Irf8-/- MΦs treated with IFNγ overnight followed by TLR ligands for 8 h was inspected by transmission electron microscopy. The bracketed region in the left panel was enlarged in the right panel. Arrows indicate autophagic vacuoles. The scale bar: 0.5 μm.(c) Reduced LC3I to LC3II conversion in Irf8-/- MΦs. WT and Irf8-/- MΦs were treated with IFNγ/TLR as above with bafilomycin A1 (200 nM) treatment for the final 2 h. Immunoblot analysis was performed with 10 μg of extracts with β-Tubulin as a control. Right panel: The amounts of LC3II in three independent samples were quantified using the ImageJ software. *p-value ≤0.05 and **p-value ≤0.01(Student's t-test). See Supplementary Fig. 3b for LC3 amount in the absence of bafilomycin A1.(d) Membrane bound LC3 in WT and Irf8-/- MΦs treated with IFNγ/TLR was detected by flow cytometry. Bafilomycin A1 (200 nM) was added for the final 2 h and. The histogram is a representative of three independent experiments. BA1: Bafilomycin A1. See Supplementary Fig. 3c for LC3 amount in the absence of bafilomycin A1.(e) Immunoblot detection of ATG5-ATG12 conjugate. WT and Irf8-/- MΦs were treated as above and immunoblot detection of ATG5-ATG12 conjugate proteins was performed. Ten microgram of extracts was tested with antibody against ATG5 or β-Tubulin.

Mentions: To test whether defective gene expression in Irf8-/- MΦs affects autophagic functions, we next examined autophagosome formation in MΦs expressing mCherry-EGFP-LC3B (Fig. 3a). This vector detects acid sensitive (EGFP) and resistant (mCherry) LC3, enabling us to assess the formation of autophagosomes and the subsequent fusion with lysosomes34. Before stimulation, GFP and mCherry signals were diffusely distributed over the cytoplasm both in WT and Irf8-/- MΦs. After IFNγ/TLR stimulation, GFP and mCherry signals relocalized to form prominent punctate structures representing autophagosomal vesicles in WT cells (see arrows in Fig. 3a). In contrast, few fluorescent vesicles were seen in Irf8-/- MΦs. Quantification in Fig. 3a (lower panel) confirmed that the number of cells with fluorescent vesicles was much fewer in Irf8-/- MΦs. Deficiency in Irf8-/- MΦs to form LC3 vesicles was also evident with endogenous LC3 (Supplementary Fig. 3a). Electron microscopy analysis additionally showed a noticeable increase in autophagic vesicles in stimulated WT MΦs, but not in Irf8-/- MΦs (Fig. 3b, right panel)35. Immunoblot analysis was performed to assess phosphatidylethalamine conjugation of LC3 as detected by changes in LC3I and LC3II levels36. Cells were treated with bafilomycin A1 to block fusion of autophagosomes to lysosomes36. Data in Fig. 3c (right panel) showed the amounts of LC3II increased in WT MΦs after IFNγ/TLR stimulation. LC3II levels were significantly lower in Irf8-/- MΦs before stimulation and did not measurably increase after simulation, as confirmed by quantification (Fig. 3c, right panel). Flow cytometric analysis to detect membrane bound LC3 further validate these result, in that LC3 signals increased after stimulation in WT MΦs. However, LC3 levels were lower in Irf8-/- MΦs before stimulation and remained low after stimulation (Fig. 3d)37. To ascertain whether IRF8 has a role in autophagosome-lysosome fusion and lysosomal clearance, immunoblot and flow cytometry assays were performed in the absence of bafilomycin A1 (Supplementary Fig. 3b,c)36. In WT MΦs, the amounts of LC3 hardly increased after stimulation, suggesting lysosomal turnover of LC3. On the other hand, LC3 levels were again lower in Irf8-/- MΦs and the amounts were unchanged after stimulation. These data support the notion that IRF8 plays a role in autophagosome formation, and subsequent autophagolysosome formation and function in IFNγ/TLR induced autophagy. Further supporting the role of IRF8 in autophagosome formation, the amounts of Atg5-Atg12 complex increased in WT MΦs, but not in Irf8-/- MΦs upon IFNγ/TLR stimulation (Fig. 3e).


IRF8 directs stress-induced autophagy in macrophages and promotes clearance of Listeria monocytogenes.

Gupta M, Shin DM, Ramakrishna L, Goussetis DJ, Platanias LC, Xiong H, Morse HC, Ozato K - Nat Commun (2015)

Defective autophagosome formation in Irf8-/- MΦs(a) LC3 vesicles were visualized in WT and Irf8-/- MΦs expressing mCherry-EGFP-LC3 vector, without (UT) or with IFNγ/TLR stimulation for 8 h. Bafilomycin A1 (200 nM) was added for final 2 h. Cells were counterstained for DNA (blue). The scale bar: 20 μm. Below: The number of cells with more than five mCherry-positive vesicles was counted by microscopic inspection of more than 200 cells. The values represent the percentage of cells with fluorescent vesicles. **p-value ≤0.01 (Student's t-test). See Supplementary Fig. 3a for endogenous staining of LC3.(b) WT and Irf8-/- MΦs treated with IFNγ overnight followed by TLR ligands for 8 h was inspected by transmission electron microscopy. The bracketed region in the left panel was enlarged in the right panel. Arrows indicate autophagic vacuoles. The scale bar: 0.5 μm.(c) Reduced LC3I to LC3II conversion in Irf8-/- MΦs. WT and Irf8-/- MΦs were treated with IFNγ/TLR as above with bafilomycin A1 (200 nM) treatment for the final 2 h. Immunoblot analysis was performed with 10 μg of extracts with β-Tubulin as a control. Right panel: The amounts of LC3II in three independent samples were quantified using the ImageJ software. *p-value ≤0.05 and **p-value ≤0.01(Student's t-test). See Supplementary Fig. 3b for LC3 amount in the absence of bafilomycin A1.(d) Membrane bound LC3 in WT and Irf8-/- MΦs treated with IFNγ/TLR was detected by flow cytometry. Bafilomycin A1 (200 nM) was added for the final 2 h and. The histogram is a representative of three independent experiments. BA1: Bafilomycin A1. See Supplementary Fig. 3c for LC3 amount in the absence of bafilomycin A1.(e) Immunoblot detection of ATG5-ATG12 conjugate. WT and Irf8-/- MΦs were treated as above and immunoblot detection of ATG5-ATG12 conjugate proteins was performed. Ten microgram of extracts was tested with antibody against ATG5 or β-Tubulin.
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Figure 3: Defective autophagosome formation in Irf8-/- MΦs(a) LC3 vesicles were visualized in WT and Irf8-/- MΦs expressing mCherry-EGFP-LC3 vector, without (UT) or with IFNγ/TLR stimulation for 8 h. Bafilomycin A1 (200 nM) was added for final 2 h. Cells were counterstained for DNA (blue). The scale bar: 20 μm. Below: The number of cells with more than five mCherry-positive vesicles was counted by microscopic inspection of more than 200 cells. The values represent the percentage of cells with fluorescent vesicles. **p-value ≤0.01 (Student's t-test). See Supplementary Fig. 3a for endogenous staining of LC3.(b) WT and Irf8-/- MΦs treated with IFNγ overnight followed by TLR ligands for 8 h was inspected by transmission electron microscopy. The bracketed region in the left panel was enlarged in the right panel. Arrows indicate autophagic vacuoles. The scale bar: 0.5 μm.(c) Reduced LC3I to LC3II conversion in Irf8-/- MΦs. WT and Irf8-/- MΦs were treated with IFNγ/TLR as above with bafilomycin A1 (200 nM) treatment for the final 2 h. Immunoblot analysis was performed with 10 μg of extracts with β-Tubulin as a control. Right panel: The amounts of LC3II in three independent samples were quantified using the ImageJ software. *p-value ≤0.05 and **p-value ≤0.01(Student's t-test). See Supplementary Fig. 3b for LC3 amount in the absence of bafilomycin A1.(d) Membrane bound LC3 in WT and Irf8-/- MΦs treated with IFNγ/TLR was detected by flow cytometry. Bafilomycin A1 (200 nM) was added for the final 2 h and. The histogram is a representative of three independent experiments. BA1: Bafilomycin A1. See Supplementary Fig. 3c for LC3 amount in the absence of bafilomycin A1.(e) Immunoblot detection of ATG5-ATG12 conjugate. WT and Irf8-/- MΦs were treated as above and immunoblot detection of ATG5-ATG12 conjugate proteins was performed. Ten microgram of extracts was tested with antibody against ATG5 or β-Tubulin.
Mentions: To test whether defective gene expression in Irf8-/- MΦs affects autophagic functions, we next examined autophagosome formation in MΦs expressing mCherry-EGFP-LC3B (Fig. 3a). This vector detects acid sensitive (EGFP) and resistant (mCherry) LC3, enabling us to assess the formation of autophagosomes and the subsequent fusion with lysosomes34. Before stimulation, GFP and mCherry signals were diffusely distributed over the cytoplasm both in WT and Irf8-/- MΦs. After IFNγ/TLR stimulation, GFP and mCherry signals relocalized to form prominent punctate structures representing autophagosomal vesicles in WT cells (see arrows in Fig. 3a). In contrast, few fluorescent vesicles were seen in Irf8-/- MΦs. Quantification in Fig. 3a (lower panel) confirmed that the number of cells with fluorescent vesicles was much fewer in Irf8-/- MΦs. Deficiency in Irf8-/- MΦs to form LC3 vesicles was also evident with endogenous LC3 (Supplementary Fig. 3a). Electron microscopy analysis additionally showed a noticeable increase in autophagic vesicles in stimulated WT MΦs, but not in Irf8-/- MΦs (Fig. 3b, right panel)35. Immunoblot analysis was performed to assess phosphatidylethalamine conjugation of LC3 as detected by changes in LC3I and LC3II levels36. Cells were treated with bafilomycin A1 to block fusion of autophagosomes to lysosomes36. Data in Fig. 3c (right panel) showed the amounts of LC3II increased in WT MΦs after IFNγ/TLR stimulation. LC3II levels were significantly lower in Irf8-/- MΦs before stimulation and did not measurably increase after simulation, as confirmed by quantification (Fig. 3c, right panel). Flow cytometric analysis to detect membrane bound LC3 further validate these result, in that LC3 signals increased after stimulation in WT MΦs. However, LC3 levels were lower in Irf8-/- MΦs before stimulation and remained low after stimulation (Fig. 3d)37. To ascertain whether IRF8 has a role in autophagosome-lysosome fusion and lysosomal clearance, immunoblot and flow cytometry assays were performed in the absence of bafilomycin A1 (Supplementary Fig. 3b,c)36. In WT MΦs, the amounts of LC3 hardly increased after stimulation, suggesting lysosomal turnover of LC3. On the other hand, LC3 levels were again lower in Irf8-/- MΦs and the amounts were unchanged after stimulation. These data support the notion that IRF8 plays a role in autophagosome formation, and subsequent autophagolysosome formation and function in IFNγ/TLR induced autophagy. Further supporting the role of IRF8 in autophagosome formation, the amounts of Atg5-Atg12 complex increased in WT MΦs, but not in Irf8-/- MΦs upon IFNγ/TLR stimulation (Fig. 3e).

Bottom Line: Autophagy, activated by many stresses, plays a critical role in innate immune responses.Here we show that interferon regulatory factor 8 (IRF8) is required for the expression of autophagy-related genes in dendritic cells.IRF8 directly activates many genes involved in various steps of autophagy, promoting autophagosome formation and lysosomal fusion.

View Article: PubMed Central - PubMed

Affiliation: Program in Genomics of Differentiation, NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
Autophagy, activated by many stresses, plays a critical role in innate immune responses. Here we show that interferon regulatory factor 8 (IRF8) is required for the expression of autophagy-related genes in dendritic cells. Furthermore in macrophages, IRF8 is induced by multiple autophagy-inducing stresses, including IFNγ and Toll-like receptor stimulation, bacterial infection, starvation and by macrophage colony-stimulating factor. IRF8 directly activates many genes involved in various steps of autophagy, promoting autophagosome formation and lysosomal fusion. Consequently, Irf8(-/-) macrophages are deficient in autophagic activity, and excessively accumulate SQSTM1 and ubiquitin-bound proteins. We show that clearance of Listeria monocytogenes in macrophages requires IRF8-dependent activation of autophagy genes and subsequent autophagic capturing and degradation of Listeria antigens. These processes are defective in Irf8(-/-) macrophages where uninhibited bacterial growth ensues. Together these data suggest that IRF8 is a major autophagy regulator in macrophages, essential for macrophage maturation, survival and innate immune responses.

Show MeSH
Related in: MedlinePlus