Limits...
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.

Show MeSH

Related in: MedlinePlus

IRF8 is required for autophagic clearance of Listeria(a) WT and Irf8-/- MΦs (106) were infected with Listeria (5×107) and bacterial transcripts were detected by qRT-PCR, normalized by Gapdh. Data represent the average of three independent experiments. p-value ≤ 0.05 (Student's t-test).(b) Autophagy gene expression in indicated MΦs was detected as above and normalized to those in uninfected WT MΦs. Data represent the average of three independent experiments. p-value ≤ 0.05 (Student's t-test).(c) Bacterial yields tested by the colony forming unit (cfu) assay. Some WT MΦs were treated with bafilomycin A1 (200 nM). Values represent the average of three determinations +/- S.D.(d) Immunoblot analysis of LC3 and SQSTM1 in Listeria infected WT and Irf8-/- MΦs in the presence of bafilomycin A1 (200 nM). Below: Amounts of LC3 and SQSTM1 from three independent samples were quantified by ImageJ software. **p-value ≤0.01(Student's t-test). See Supplementary Fig. 7a for LC3 and SQSTM1 amounts in the absence of bafilomycin A1.(e) Distribution of Listeria antigens and LC3 after 36 h of Listeria infection in immunostaining. Arrow heads: LC3-associated Listeria. The scale bar: 20 μm. Below: The percentage of cells showing co-localization of LC3 and Listeria antigens. Values represent the average of three independent experiments +/- S.D with **p ≤0.01 (Student's t-test).(f) Bacterial yields in MΦs infected with Listeria for 24 h and treated with Act D (2 μg/ml). Data represent the average of three determinations +/- S.D. See Supplementary Fig. 8 for details of Act D effects.(g)Irf8-/- MΦs transduced with Irf8 vector were infected with Listeria for 36 h and autophagy gene expression was detected as above. The numbers represent transcript levels normalized by those with empty vector. Irf8 transcripts were normalized by gapdh. Values are the average of three experiments. p-value ≤0.05 (Student's t-test). Hprt: a negative control.(h) Left and middle: Irf8-/- MΦs transduced as above were tested for LC3II using GAPDH as a control. The amounts of LC3II in two independent experiments were quantified using ImageJ software. **p-value ≤0.01(Student's t-test). Right: Irf8-/- MΦs expressing mCherry-EGFP-LC3 were transduced as above and infected with Listeria for 36 h and fluorescent LC3 signals was visualized by confocal microscopy. See Supplementary Fig. 7b for microscopy image with controls.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4363081&req=5

Figure 6: IRF8 is required for autophagic clearance of Listeria(a) WT and Irf8-/- MΦs (106) were infected with Listeria (5×107) and bacterial transcripts were detected by qRT-PCR, normalized by Gapdh. Data represent the average of three independent experiments. p-value ≤ 0.05 (Student's t-test).(b) Autophagy gene expression in indicated MΦs was detected as above and normalized to those in uninfected WT MΦs. Data represent the average of three independent experiments. p-value ≤ 0.05 (Student's t-test).(c) Bacterial yields tested by the colony forming unit (cfu) assay. Some WT MΦs were treated with bafilomycin A1 (200 nM). Values represent the average of three determinations +/- S.D.(d) Immunoblot analysis of LC3 and SQSTM1 in Listeria infected WT and Irf8-/- MΦs in the presence of bafilomycin A1 (200 nM). Below: Amounts of LC3 and SQSTM1 from three independent samples were quantified by ImageJ software. **p-value ≤0.01(Student's t-test). See Supplementary Fig. 7a for LC3 and SQSTM1 amounts in the absence of bafilomycin A1.(e) Distribution of Listeria antigens and LC3 after 36 h of Listeria infection in immunostaining. Arrow heads: LC3-associated Listeria. The scale bar: 20 μm. Below: The percentage of cells showing co-localization of LC3 and Listeria antigens. Values represent the average of three independent experiments +/- S.D with **p ≤0.01 (Student's t-test).(f) Bacterial yields in MΦs infected with Listeria for 24 h and treated with Act D (2 μg/ml). Data represent the average of three determinations +/- S.D. See Supplementary Fig. 8 for details of Act D effects.(g)Irf8-/- MΦs transduced with Irf8 vector were infected with Listeria for 36 h and autophagy gene expression was detected as above. The numbers represent transcript levels normalized by those with empty vector. Irf8 transcripts were normalized by gapdh. Values are the average of three experiments. p-value ≤0.05 (Student's t-test). Hprt: a negative control.(h) Left and middle: Irf8-/- MΦs transduced as above were tested for LC3II using GAPDH as a control. The amounts of LC3II in two independent experiments were quantified using ImageJ software. **p-value ≤0.01(Student's t-test). Right: Irf8-/- MΦs expressing mCherry-EGFP-LC3 were transduced as above and infected with Listeria for 36 h and fluorescent LC3 signals was visualized by confocal microscopy. See Supplementary Fig. 7b for microscopy image with controls.

Mentions: Autophagy is a major mechanism by which MΦs eliminate intracellular bacteria, such as M. tuberculosis, Salmonella, and Listeria6,10,41. Previous studies showed that IRF8 is essential for controlling infection of the above bacteria22-26. However, it has not been clear whether IRF8 employs autophagy to confer protection. We first examined whether Listeria infection activates autophagy genes in MΦs: Listeria monocytogenes is a food-born pathogen that causes listeriosis, and widely studied in mouse models42. qRT-PCR data in Fig. 6a showed that expression of Listeria transcripts rose sharply in Irf8-/- MΦs during 48 h of infection, while remained very low in WT MΦs, including ActA shown to bypass host cell autophagy41. On the other hand, Listeria infection markedly increased expression of many autophagy genes in WT MΦs, in some cases by nearly 100-folds (Fig. 6b). Among induced genes was Nod1, important for autophagy-mediated control of Listeria43. Interestingly, upon Listeria infection, Irf8 expression was also dramatically increased. In contrast, none of these autophagy genes were induced in Irf8-/- MΦs at comparable levels (Fig. 6b). Accordingly, while WT MΦs restricted bacterial growth to a minimum, Irf8-/- MΦs relented to the logarithmic growth of Listeria, as verified by bacterial accumulation in the cytoplasm (Fig. 6c). Further supporting IRF8 dependent autophagic activation, LC3II levels increased during Listeria infection in WT MΦs but not in Irf8-/- MΦs, as observed in the presence of bafilomycin A1 (Fig. 6d). In contrast, LC3II amounts remained much lower in Irf8-/- MΦs in during infection. In the absence of bafilomycin A1, LC3II amounts increased at 24 h, then gradually decreased afterwards up to 48 h (Supplementary Fig. 7a) in WT MΦs. In Irf8-/- MΦs, LC3II amounts also increased at 24 h, but remained high throughout 48 h of infection, suggesting inefficient LC3 turnover in lysosomes36,44. In agreement, SQSTM1 and SQSTM1-positive proteins accumulated in Irf8-/- MΦs in greater amounts than WT cells with and without bafilomycin A1 (Fig. 6d, Supplementary Fig. 7a). Together, these results reinforce the view that during Listeria infection, IRF8 plays a major role in promoting both autophagosome formation and the subsequent autolysosomal functions. Importantly, immunostaining analysis in Fig. 6e revealed that Listeria antigens co-localized with LC3 and formed autophagosomal vesicles in WT cells, indicative of autophagic capturing of bacterial antigens. However, the Listeria antigens were more abundant in Irf8-/- MΦs than in WT cells and the co-localization with LC3 was hardly observed (Fig. 6e). We also found that blocking of autophagosome-lysosome fusion by bafilomycin A1 led to logarithmic growth of Listeria in WT MΦs, comparable to that in Irf8-/- MΦs, supporting autophagic control of Listeria infection (Fig. 6c). To ascertain whether autophagic control of Listeria relies on de novo transcription of autophagy genes, infected WT and Irf8-/- MΦs were briefly treated with Actinomycin D (Act D) and tested for bacterial growth. As shown in Fig. 6f, bacterial counts sharply increased after 2 h of Act D treatment both in WT and Irf8-/- MΦs. Act D also inhibited autophagosome formation and expression of autophagy genes upon IFNγ/TLR stimulation (Supplementary Fig. 8). These results are analogous to the previous report where muscle cell autophagy depends on FOXO3 mediated transcription of autophagy genes17.


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)

IRF8 is required for autophagic clearance of Listeria(a) WT and Irf8-/- MΦs (106) were infected with Listeria (5×107) and bacterial transcripts were detected by qRT-PCR, normalized by Gapdh. Data represent the average of three independent experiments. p-value ≤ 0.05 (Student's t-test).(b) Autophagy gene expression in indicated MΦs was detected as above and normalized to those in uninfected WT MΦs. Data represent the average of three independent experiments. p-value ≤ 0.05 (Student's t-test).(c) Bacterial yields tested by the colony forming unit (cfu) assay. Some WT MΦs were treated with bafilomycin A1 (200 nM). Values represent the average of three determinations +/- S.D.(d) Immunoblot analysis of LC3 and SQSTM1 in Listeria infected WT and Irf8-/- MΦs in the presence of bafilomycin A1 (200 nM). Below: Amounts of LC3 and SQSTM1 from three independent samples were quantified by ImageJ software. **p-value ≤0.01(Student's t-test). See Supplementary Fig. 7a for LC3 and SQSTM1 amounts in the absence of bafilomycin A1.(e) Distribution of Listeria antigens and LC3 after 36 h of Listeria infection in immunostaining. Arrow heads: LC3-associated Listeria. The scale bar: 20 μm. Below: The percentage of cells showing co-localization of LC3 and Listeria antigens. Values represent the average of three independent experiments +/- S.D with **p ≤0.01 (Student's t-test).(f) Bacterial yields in MΦs infected with Listeria for 24 h and treated with Act D (2 μg/ml). Data represent the average of three determinations +/- S.D. See Supplementary Fig. 8 for details of Act D effects.(g)Irf8-/- MΦs transduced with Irf8 vector were infected with Listeria for 36 h and autophagy gene expression was detected as above. The numbers represent transcript levels normalized by those with empty vector. Irf8 transcripts were normalized by gapdh. Values are the average of three experiments. p-value ≤0.05 (Student's t-test). Hprt: a negative control.(h) Left and middle: Irf8-/- MΦs transduced as above were tested for LC3II using GAPDH as a control. The amounts of LC3II in two independent experiments were quantified using ImageJ software. **p-value ≤0.01(Student's t-test). Right: Irf8-/- MΦs expressing mCherry-EGFP-LC3 were transduced as above and infected with Listeria for 36 h and fluorescent LC3 signals was visualized by confocal microscopy. See Supplementary Fig. 7b for microscopy image with controls.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4363081&req=5

Figure 6: IRF8 is required for autophagic clearance of Listeria(a) WT and Irf8-/- MΦs (106) were infected with Listeria (5×107) and bacterial transcripts were detected by qRT-PCR, normalized by Gapdh. Data represent the average of three independent experiments. p-value ≤ 0.05 (Student's t-test).(b) Autophagy gene expression in indicated MΦs was detected as above and normalized to those in uninfected WT MΦs. Data represent the average of three independent experiments. p-value ≤ 0.05 (Student's t-test).(c) Bacterial yields tested by the colony forming unit (cfu) assay. Some WT MΦs were treated with bafilomycin A1 (200 nM). Values represent the average of three determinations +/- S.D.(d) Immunoblot analysis of LC3 and SQSTM1 in Listeria infected WT and Irf8-/- MΦs in the presence of bafilomycin A1 (200 nM). Below: Amounts of LC3 and SQSTM1 from three independent samples were quantified by ImageJ software. **p-value ≤0.01(Student's t-test). See Supplementary Fig. 7a for LC3 and SQSTM1 amounts in the absence of bafilomycin A1.(e) Distribution of Listeria antigens and LC3 after 36 h of Listeria infection in immunostaining. Arrow heads: LC3-associated Listeria. The scale bar: 20 μm. Below: The percentage of cells showing co-localization of LC3 and Listeria antigens. Values represent the average of three independent experiments +/- S.D with **p ≤0.01 (Student's t-test).(f) Bacterial yields in MΦs infected with Listeria for 24 h and treated with Act D (2 μg/ml). Data represent the average of three determinations +/- S.D. See Supplementary Fig. 8 for details of Act D effects.(g)Irf8-/- MΦs transduced with Irf8 vector were infected with Listeria for 36 h and autophagy gene expression was detected as above. The numbers represent transcript levels normalized by those with empty vector. Irf8 transcripts were normalized by gapdh. Values are the average of three experiments. p-value ≤0.05 (Student's t-test). Hprt: a negative control.(h) Left and middle: Irf8-/- MΦs transduced as above were tested for LC3II using GAPDH as a control. The amounts of LC3II in two independent experiments were quantified using ImageJ software. **p-value ≤0.01(Student's t-test). Right: Irf8-/- MΦs expressing mCherry-EGFP-LC3 were transduced as above and infected with Listeria for 36 h and fluorescent LC3 signals was visualized by confocal microscopy. See Supplementary Fig. 7b for microscopy image with controls.
Mentions: Autophagy is a major mechanism by which MΦs eliminate intracellular bacteria, such as M. tuberculosis, Salmonella, and Listeria6,10,41. Previous studies showed that IRF8 is essential for controlling infection of the above bacteria22-26. However, it has not been clear whether IRF8 employs autophagy to confer protection. We first examined whether Listeria infection activates autophagy genes in MΦs: Listeria monocytogenes is a food-born pathogen that causes listeriosis, and widely studied in mouse models42. qRT-PCR data in Fig. 6a showed that expression of Listeria transcripts rose sharply in Irf8-/- MΦs during 48 h of infection, while remained very low in WT MΦs, including ActA shown to bypass host cell autophagy41. On the other hand, Listeria infection markedly increased expression of many autophagy genes in WT MΦs, in some cases by nearly 100-folds (Fig. 6b). Among induced genes was Nod1, important for autophagy-mediated control of Listeria43. Interestingly, upon Listeria infection, Irf8 expression was also dramatically increased. In contrast, none of these autophagy genes were induced in Irf8-/- MΦs at comparable levels (Fig. 6b). Accordingly, while WT MΦs restricted bacterial growth to a minimum, Irf8-/- MΦs relented to the logarithmic growth of Listeria, as verified by bacterial accumulation in the cytoplasm (Fig. 6c). Further supporting IRF8 dependent autophagic activation, LC3II levels increased during Listeria infection in WT MΦs but not in Irf8-/- MΦs, as observed in the presence of bafilomycin A1 (Fig. 6d). In contrast, LC3II amounts remained much lower in Irf8-/- MΦs in during infection. In the absence of bafilomycin A1, LC3II amounts increased at 24 h, then gradually decreased afterwards up to 48 h (Supplementary Fig. 7a) in WT MΦs. In Irf8-/- MΦs, LC3II amounts also increased at 24 h, but remained high throughout 48 h of infection, suggesting inefficient LC3 turnover in lysosomes36,44. In agreement, SQSTM1 and SQSTM1-positive proteins accumulated in Irf8-/- MΦs in greater amounts than WT cells with and without bafilomycin A1 (Fig. 6d, Supplementary Fig. 7a). Together, these results reinforce the view that during Listeria infection, IRF8 plays a major role in promoting both autophagosome formation and the subsequent autolysosomal functions. Importantly, immunostaining analysis in Fig. 6e revealed that Listeria antigens co-localized with LC3 and formed autophagosomal vesicles in WT cells, indicative of autophagic capturing of bacterial antigens. However, the Listeria antigens were more abundant in Irf8-/- MΦs than in WT cells and the co-localization with LC3 was hardly observed (Fig. 6e). We also found that blocking of autophagosome-lysosome fusion by bafilomycin A1 led to logarithmic growth of Listeria in WT MΦs, comparable to that in Irf8-/- MΦs, supporting autophagic control of Listeria infection (Fig. 6c). To ascertain whether autophagic control of Listeria relies on de novo transcription of autophagy genes, infected WT and Irf8-/- MΦs were briefly treated with Actinomycin D (Act D) and tested for bacterial growth. As shown in Fig. 6f, bacterial counts sharply increased after 2 h of Act D treatment both in WT and Irf8-/- MΦs. Act D also inhibited autophagosome formation and expression of autophagy genes upon IFNγ/TLR stimulation (Supplementary Fig. 8). These results are analogous to the previous report where muscle cell autophagy depends on FOXO3 mediated transcription of autophagy genes17.

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