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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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Microarray analyses reveal a role of IRF8 in autophagy(a) The Venn diagrams depict the number of genes positively and negatively regulated by IRF8 in untreated (UT) DCs and those treated with TLR ligands for 6 h identified by microarray analysis. Overlapped regions represent the number of genes positively or negatively regulated both in untreated and TLR stimulated DCs. See GO classification in Supplementary Fig. 1a,b.(b) IRF8 dependent autophagy genes identified by microarray analysis. Differentially expressed genes were identified by one-way ANOVA (p-value≤0.05 & fold change ≥2). Color gradients indicate average signal intensities of genes in log2 scale. Normalization was performed by GeneChip Operating Software (GCOS) and Expression Console software's. relative to the Untreated (UT).(c) qRT-PCR analysis of IRF8 dependent autophagy genes in DCs. WT and Irf8-/- DCs were stimulated with the TLR ligands for 6 h. The numbers represent transcript levels normalized by gapdh levels. Data represents the average of three independent assays. p-value ≤0.01., Student's t-test.(d) qRT-PCR analysis of IRF8 dependent autophagy genes in MΦs. WT and Irf8-/- MΦs were treated overnight with IFNγ and stimulated with TLR ligands for the indicated times. The number in each box represents transcript levels normalized by the value of untreated WT cells. Values are the average of three independent assays. p-value ≤0.01(Student's t-test). See autophagy genes not affected by IRF8 in Supplementary Fig. 2.(e)Irf8-/- MΦs were transduced with pMSCV retroviral vector containing WT Irf8 or mutant Irf8 (K79E), and stimulated with IFNγ/TLR for 8 h. Relative expression of indicated autophagy genes was detected by qRT-PCR. The numbers represent transcript levels normalized by those of cells transduced with empty vector. Irf8 expression was normalized by gapdh. Values are the average of three experiments. p-value ≤0.05 (Student's t-test). Il12b and Hprt were tested as controls.
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Figure 1: Microarray analyses reveal a role of IRF8 in autophagy(a) The Venn diagrams depict the number of genes positively and negatively regulated by IRF8 in untreated (UT) DCs and those treated with TLR ligands for 6 h identified by microarray analysis. Overlapped regions represent the number of genes positively or negatively regulated both in untreated and TLR stimulated DCs. See GO classification in Supplementary Fig. 1a,b.(b) IRF8 dependent autophagy genes identified by microarray analysis. Differentially expressed genes were identified by one-way ANOVA (p-value≤0.05 & fold change ≥2). Color gradients indicate average signal intensities of genes in log2 scale. Normalization was performed by GeneChip Operating Software (GCOS) and Expression Console software's. relative to the Untreated (UT).(c) qRT-PCR analysis of IRF8 dependent autophagy genes in DCs. WT and Irf8-/- DCs were stimulated with the TLR ligands for 6 h. The numbers represent transcript levels normalized by gapdh levels. Data represents the average of three independent assays. p-value ≤0.01., Student's t-test.(d) qRT-PCR analysis of IRF8 dependent autophagy genes in MΦs. WT and Irf8-/- MΦs were treated overnight with IFNγ and stimulated with TLR ligands for the indicated times. The number in each box represents transcript levels normalized by the value of untreated WT cells. Values are the average of three independent assays. p-value ≤0.01(Student's t-test). See autophagy genes not affected by IRF8 in Supplementary Fig. 2.(e)Irf8-/- MΦs were transduced with pMSCV retroviral vector containing WT Irf8 or mutant Irf8 (K79E), and stimulated with IFNγ/TLR for 8 h. Relative expression of indicated autophagy genes was detected by qRT-PCR. The numbers represent transcript levels normalized by those of cells transduced with empty vector. Irf8 expression was normalized by gapdh. Values are the average of three experiments. p-value ≤0.05 (Student's t-test). Il12b and Hprt were tested as controls.

Mentions: Previous genome-wide studies reported that IRF8 regulates more than 1,500 genes in monocytes, MΦs and B cells21,27,28. To gain genome-wide information on IRF8 in DCs, we performed microarray analyses with bone marrow (BM) derived DCs from wild type (WT) and Irf8-/- mice upon stimulation by TLR ligands, LPS and CpG. With a cut off line of >2× with p≤0.05 (identified by one-way ANOVA), 326 and 713 genes were expressed higher in WT DCs than in Irf8-/- DCs in untreated (UT) and TLR stimulated DCs, respectively (Fig. 1a, left). Whereas, expression of 350 and 648 genes was lower in WT DCs than in Irf8-/- DCs (Fig. 1a, right). Thus, IRF8 regulates many constitutive and TLR-stimulated genes in DCs either positively or negatively, as reported before for other cell types21,27,28. Gene ontology (GO) analysis of positively regulated genes showed significant enrichment for immune system processes, inflammatory responses, lysosome functions, while genes negatively regulated by IRF8 were enriched with cell cycle, cell division and DNA replication (Supplementary Fig. 1a,b). A large number of TLR-stimulated genes were up regulated by IRF8, consistent with previous reports that IRF8 is critical for TLR activation of DCs (Supplementary Fig. 1c and Supplementary Table 1)18,20,29. Eleven percent of those were found in the Interferome, confirming a functional link between IRF8 and IFN related regulation (http://interferome.org/)30 (Supplementary Fig. 1d).


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)

Microarray analyses reveal a role of IRF8 in autophagy(a) The Venn diagrams depict the number of genes positively and negatively regulated by IRF8 in untreated (UT) DCs and those treated with TLR ligands for 6 h identified by microarray analysis. Overlapped regions represent the number of genes positively or negatively regulated both in untreated and TLR stimulated DCs. See GO classification in Supplementary Fig. 1a,b.(b) IRF8 dependent autophagy genes identified by microarray analysis. Differentially expressed genes were identified by one-way ANOVA (p-value≤0.05 & fold change ≥2). Color gradients indicate average signal intensities of genes in log2 scale. Normalization was performed by GeneChip Operating Software (GCOS) and Expression Console software's. relative to the Untreated (UT).(c) qRT-PCR analysis of IRF8 dependent autophagy genes in DCs. WT and Irf8-/- DCs were stimulated with the TLR ligands for 6 h. The numbers represent transcript levels normalized by gapdh levels. Data represents the average of three independent assays. p-value ≤0.01., Student's t-test.(d) qRT-PCR analysis of IRF8 dependent autophagy genes in MΦs. WT and Irf8-/- MΦs were treated overnight with IFNγ and stimulated with TLR ligands for the indicated times. The number in each box represents transcript levels normalized by the value of untreated WT cells. Values are the average of three independent assays. p-value ≤0.01(Student's t-test). See autophagy genes not affected by IRF8 in Supplementary Fig. 2.(e)Irf8-/- MΦs were transduced with pMSCV retroviral vector containing WT Irf8 or mutant Irf8 (K79E), and stimulated with IFNγ/TLR for 8 h. Relative expression of indicated autophagy genes was detected by qRT-PCR. The numbers represent transcript levels normalized by those of cells transduced with empty vector. Irf8 expression was normalized by gapdh. Values are the average of three experiments. p-value ≤0.05 (Student's t-test). Il12b and Hprt were tested as controls.
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Figure 1: Microarray analyses reveal a role of IRF8 in autophagy(a) The Venn diagrams depict the number of genes positively and negatively regulated by IRF8 in untreated (UT) DCs and those treated with TLR ligands for 6 h identified by microarray analysis. Overlapped regions represent the number of genes positively or negatively regulated both in untreated and TLR stimulated DCs. See GO classification in Supplementary Fig. 1a,b.(b) IRF8 dependent autophagy genes identified by microarray analysis. Differentially expressed genes were identified by one-way ANOVA (p-value≤0.05 & fold change ≥2). Color gradients indicate average signal intensities of genes in log2 scale. Normalization was performed by GeneChip Operating Software (GCOS) and Expression Console software's. relative to the Untreated (UT).(c) qRT-PCR analysis of IRF8 dependent autophagy genes in DCs. WT and Irf8-/- DCs were stimulated with the TLR ligands for 6 h. The numbers represent transcript levels normalized by gapdh levels. Data represents the average of three independent assays. p-value ≤0.01., Student's t-test.(d) qRT-PCR analysis of IRF8 dependent autophagy genes in MΦs. WT and Irf8-/- MΦs were treated overnight with IFNγ and stimulated with TLR ligands for the indicated times. The number in each box represents transcript levels normalized by the value of untreated WT cells. Values are the average of three independent assays. p-value ≤0.01(Student's t-test). See autophagy genes not affected by IRF8 in Supplementary Fig. 2.(e)Irf8-/- MΦs were transduced with pMSCV retroviral vector containing WT Irf8 or mutant Irf8 (K79E), and stimulated with IFNγ/TLR for 8 h. Relative expression of indicated autophagy genes was detected by qRT-PCR. The numbers represent transcript levels normalized by those of cells transduced with empty vector. Irf8 expression was normalized by gapdh. Values are the average of three experiments. p-value ≤0.05 (Student's t-test). Il12b and Hprt were tested as controls.
Mentions: Previous genome-wide studies reported that IRF8 regulates more than 1,500 genes in monocytes, MΦs and B cells21,27,28. To gain genome-wide information on IRF8 in DCs, we performed microarray analyses with bone marrow (BM) derived DCs from wild type (WT) and Irf8-/- mice upon stimulation by TLR ligands, LPS and CpG. With a cut off line of >2× with p≤0.05 (identified by one-way ANOVA), 326 and 713 genes were expressed higher in WT DCs than in Irf8-/- DCs in untreated (UT) and TLR stimulated DCs, respectively (Fig. 1a, left). Whereas, expression of 350 and 648 genes was lower in WT DCs than in Irf8-/- DCs (Fig. 1a, right). Thus, IRF8 regulates many constitutive and TLR-stimulated genes in DCs either positively or negatively, as reported before for other cell types21,27,28. Gene ontology (GO) analysis of positively regulated genes showed significant enrichment for immune system processes, inflammatory responses, lysosome functions, while genes negatively regulated by IRF8 were enriched with cell cycle, cell division and DNA replication (Supplementary Fig. 1a,b). A large number of TLR-stimulated genes were up regulated by IRF8, consistent with previous reports that IRF8 is critical for TLR activation of DCs (Supplementary Fig. 1c and Supplementary Table 1)18,20,29. Eleven percent of those were found in the Interferome, confirming a functional link between IRF8 and IFN related regulation (http://interferome.org/)30 (Supplementary Fig. 1d).

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