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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: 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.

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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SQSTM1 and ubiquitin-conjugated proteins form aggregates in Irf8-/- MΦs(a) SQSTM1- and ubiquitin-positive proteins (arrow heads) in WT and Irf8-/- MΦs stimulated with IFNγ overnight and TLR ligands for 8 h were visualized by immunostaining. Bafilomycin A1 (200 nM) was added for final 2 h. Cells were counterstained for DNA (blue). Right panel: The percentage of cells with SQSTM1- and ubiquitin-positive aggregates. Data represent the average of three independent experiments +/- S.D with **p-value ≤0.01 (Student's t-test).(b) Immunoblot detection of SQSTM1- and ubiquitin-positive proteins in WT and Irf8-/- MΦs stimulated with IFNγ or IFNγ/TLR for 8 h. Bafilomycin A1 (200 nM) was added for final 2 h. Right panel: Relative amounts of ubiquitin-bound proteins. Data represent the average of three independent experiments +/- S.D with **p-value ≤ 0.01 (Student's t-test). See Supplementary Fig. 3b for immunoblot detection of SQSTM1 in the absence of bafilomycin A1.(c) Immunoblot detection of ubiquitin-bound proteins in the presence of MG132. WT and Irf8-/- MΦs were stimulated with IFNγ/TLR in the presence of 10 μM of MG132. Right panel: Relative amounts of ubiquitin bound proteins. Data represents the average of three independent experiments +/- S.D with *p-value ≤0.05 and **p-value ≤0.01 (Student's t-test).
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Figure 5: SQSTM1 and ubiquitin-conjugated proteins form aggregates in Irf8-/- MΦs(a) SQSTM1- and ubiquitin-positive proteins (arrow heads) in WT and Irf8-/- MΦs stimulated with IFNγ overnight and TLR ligands for 8 h were visualized by immunostaining. Bafilomycin A1 (200 nM) was added for final 2 h. Cells were counterstained for DNA (blue). Right panel: The percentage of cells with SQSTM1- and ubiquitin-positive aggregates. Data represent the average of three independent experiments +/- S.D with **p-value ≤0.01 (Student's t-test).(b) Immunoblot detection of SQSTM1- and ubiquitin-positive proteins in WT and Irf8-/- MΦs stimulated with IFNγ or IFNγ/TLR for 8 h. Bafilomycin A1 (200 nM) was added for final 2 h. Right panel: Relative amounts of ubiquitin-bound proteins. Data represent the average of three independent experiments +/- S.D with **p-value ≤ 0.01 (Student's t-test). See Supplementary Fig. 3b for immunoblot detection of SQSTM1 in the absence of bafilomycin A1.(c) Immunoblot detection of ubiquitin-bound proteins in the presence of MG132. WT and Irf8-/- MΦs were stimulated with IFNγ/TLR in the presence of 10 μM of MG132. Right panel: Relative amounts of ubiquitin bound proteins. Data represents the average of three independent experiments +/- S.D with *p-value ≤0.05 and **p-value ≤0.01 (Student's t-test).

Mentions: Misfolded self-proteins and invading pathogens are often conjugated to ubiquitin and escorted to autophagosomes to be degraded in lysosomes. This process is partly mediated by the ubiquitin adaptor SQSTM1 (p62)13,14. Some autophagy deficient cells form aggregates, called aggresomes or inclusion bodies that contain SQSTM1 and ubiquitin14. In light of defective autophagy, it was possible that Irf8-/- MΦs abnormally accumulate SQSTM1 and ubiquitin-containing proteins. Immunostaining of ubiquitin and SQSTM1 showed extensive deposition of SQSTM1 that co-localized with ubiquitin-positive proteins in Irf8-/- MΦs after IFNγ/TLR stimulation, in contrast to WT MΦs which showed little deposition of these proteins (Fig. 5a, left panel). Quantification of SQSTM1 and ubiquitin-positive cells confirmed the immunostaining results (Fig. 5a, right panel). Immunoblot analysis further confirmed that SQSTM1 accumulates in greater amounts in Irf8-/- than WT MΦs with and without bafilomycin A1 treatment (Fig. 5b upper panel and Supplementary Fig. 3b). In addition, the amounts of ubiquitin-positive proteins increased markedly after stimulation and to a greater extent in Irf8-/- MΦs than WT cells (Fig. 5b, left and right panels). In agreement with the increased ubiquitin-positive proteins, we previously noted that IFNγ/TLR stimulation increases ubiquitin-conjugated proteins in MΦs39. Next we tested whether the larger increase in SQSTM1 protein expression in Irf8-/- cells was due to higher Sqstm1 transcription in Irf8-/- MΦs. Relevant to this question, we previously showed that Sqstm1 mRNA expression increases after IFNγ/TLR stimulation in WT MΦs40. qRT-PCR data showed that levels of Sqstm1 mRNA were comparable in WT and Irf8-/- MΦs, indicating that SQSTM1 proteins aberrantly accumulate in Irf8-/- cells after stimulation, due to deficiency in autophagic degradation (Supplementary Fig. 5). To ascertain the role of autophagic degradation in the elimination of ubiquitin conjugated proteins, in addition to proteasome mediated degradation, immunoblot analysis was performed for cells treated with a proteasome inhibitor, MG132. As seen in Fig. 5c left and right panels, Irf8-/- MΦs accumulated greater amounts of ubiquitin-bound proteins than WT MΦs in the presence of MG132, suggesting that autophagy partly accounted for excess accumulation of SQSTM1 and ubiquitin-bound proteins in Irf8-/- MΦs. Further supporting accumulation of unprocessed proteins in Irf8-/- MΦs, MitoTracker-positive materials that co-localized with monodansylcadaverine (MDC) staining were more abundant in Irf8-/- MΦs than WT cells (Supplementary Fig. 6)36.


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)

SQSTM1 and ubiquitin-conjugated proteins form aggregates in Irf8-/- MΦs(a) SQSTM1- and ubiquitin-positive proteins (arrow heads) in WT and Irf8-/- MΦs stimulated with IFNγ overnight and TLR ligands for 8 h were visualized by immunostaining. Bafilomycin A1 (200 nM) was added for final 2 h. Cells were counterstained for DNA (blue). Right panel: The percentage of cells with SQSTM1- and ubiquitin-positive aggregates. Data represent the average of three independent experiments +/- S.D with **p-value ≤0.01 (Student's t-test).(b) Immunoblot detection of SQSTM1- and ubiquitin-positive proteins in WT and Irf8-/- MΦs stimulated with IFNγ or IFNγ/TLR for 8 h. Bafilomycin A1 (200 nM) was added for final 2 h. Right panel: Relative amounts of ubiquitin-bound proteins. Data represent the average of three independent experiments +/- S.D with **p-value ≤ 0.01 (Student's t-test). See Supplementary Fig. 3b for immunoblot detection of SQSTM1 in the absence of bafilomycin A1.(c) Immunoblot detection of ubiquitin-bound proteins in the presence of MG132. WT and Irf8-/- MΦs were stimulated with IFNγ/TLR in the presence of 10 μM of MG132. Right panel: Relative amounts of ubiquitin bound proteins. Data represents the average of three independent experiments +/- S.D with *p-value ≤0.05 and **p-value ≤0.01 (Student's t-test).
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Figure 5: SQSTM1 and ubiquitin-conjugated proteins form aggregates in Irf8-/- MΦs(a) SQSTM1- and ubiquitin-positive proteins (arrow heads) in WT and Irf8-/- MΦs stimulated with IFNγ overnight and TLR ligands for 8 h were visualized by immunostaining. Bafilomycin A1 (200 nM) was added for final 2 h. Cells were counterstained for DNA (blue). Right panel: The percentage of cells with SQSTM1- and ubiquitin-positive aggregates. Data represent the average of three independent experiments +/- S.D with **p-value ≤0.01 (Student's t-test).(b) Immunoblot detection of SQSTM1- and ubiquitin-positive proteins in WT and Irf8-/- MΦs stimulated with IFNγ or IFNγ/TLR for 8 h. Bafilomycin A1 (200 nM) was added for final 2 h. Right panel: Relative amounts of ubiquitin-bound proteins. Data represent the average of three independent experiments +/- S.D with **p-value ≤ 0.01 (Student's t-test). See Supplementary Fig. 3b for immunoblot detection of SQSTM1 in the absence of bafilomycin A1.(c) Immunoblot detection of ubiquitin-bound proteins in the presence of MG132. WT and Irf8-/- MΦs were stimulated with IFNγ/TLR in the presence of 10 μM of MG132. Right panel: Relative amounts of ubiquitin bound proteins. Data represents the average of three independent experiments +/- S.D with *p-value ≤0.05 and **p-value ≤0.01 (Student's t-test).
Mentions: Misfolded self-proteins and invading pathogens are often conjugated to ubiquitin and escorted to autophagosomes to be degraded in lysosomes. This process is partly mediated by the ubiquitin adaptor SQSTM1 (p62)13,14. Some autophagy deficient cells form aggregates, called aggresomes or inclusion bodies that contain SQSTM1 and ubiquitin14. In light of defective autophagy, it was possible that Irf8-/- MΦs abnormally accumulate SQSTM1 and ubiquitin-containing proteins. Immunostaining of ubiquitin and SQSTM1 showed extensive deposition of SQSTM1 that co-localized with ubiquitin-positive proteins in Irf8-/- MΦs after IFNγ/TLR stimulation, in contrast to WT MΦs which showed little deposition of these proteins (Fig. 5a, left panel). Quantification of SQSTM1 and ubiquitin-positive cells confirmed the immunostaining results (Fig. 5a, right panel). Immunoblot analysis further confirmed that SQSTM1 accumulates in greater amounts in Irf8-/- than WT MΦs with and without bafilomycin A1 treatment (Fig. 5b upper panel and Supplementary Fig. 3b). In addition, the amounts of ubiquitin-positive proteins increased markedly after stimulation and to a greater extent in Irf8-/- MΦs than WT cells (Fig. 5b, left and right panels). In agreement with the increased ubiquitin-positive proteins, we previously noted that IFNγ/TLR stimulation increases ubiquitin-conjugated proteins in MΦs39. Next we tested whether the larger increase in SQSTM1 protein expression in Irf8-/- cells was due to higher Sqstm1 transcription in Irf8-/- MΦs. Relevant to this question, we previously showed that Sqstm1 mRNA expression increases after IFNγ/TLR stimulation in WT MΦs40. qRT-PCR data showed that levels of Sqstm1 mRNA were comparable in WT and Irf8-/- MΦs, indicating that SQSTM1 proteins aberrantly accumulate in Irf8-/- cells after stimulation, due to deficiency in autophagic degradation (Supplementary Fig. 5). To ascertain the role of autophagic degradation in the elimination of ubiquitin conjugated proteins, in addition to proteasome mediated degradation, immunoblot analysis was performed for cells treated with a proteasome inhibitor, MG132. As seen in Fig. 5c left and right panels, Irf8-/- MΦs accumulated greater amounts of ubiquitin-bound proteins than WT MΦs in the presence of MG132, suggesting that autophagy partly accounted for excess accumulation of SQSTM1 and ubiquitin-bound proteins in Irf8-/- MΦs. Further supporting accumulation of unprocessed proteins in Irf8-/- MΦs, MitoTracker-positive materials that co-localized with monodansylcadaverine (MDC) staining were more abundant in Irf8-/- MΦs than WT cells (Supplementary Fig. 6)36.

Bottom Line: 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.

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