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Host defense against viral infection involves interferon mediated down-regulation of sterol biosynthesis.

Blanc M, Hsieh WY, Robertson KA, Watterson S, Shui G, Lacaze P, Khondoker M, Dickinson P, Sing G, Rodríguez-Martín S, Phelan P, Forster T, Strobl B, Müller M, Riemersma R, Osborne T, Wenk MR, Angulo A, Ghazal P - PLoS Biol. (2011)

Bottom Line: Experimental testing of sterol metabolite levels using lipidomic-based measurements shows a reduction in metabolic output.Altogether these results show that type 1 IFN signaling is both necessary and sufficient for reducing the sterol metabolic network activity upon infection, thereby linking the regulation of the sterol pathway with interferon anti-viral defense responses.These findings bring a new link between sterol metabolism and interferon antiviral response and support the idea of using host metabolic modifiers of innate immunity as a potential antiviral strategy.

View Article: PubMed Central - PubMed

Affiliation: Division of Pathway Medicine and Centre for Infectious Diseases, University of Edinburgh, Edinburgh, United Kingdom.

ABSTRACT
Little is known about the protective role of inflammatory processes in modulating lipid metabolism in infection. Here we report an intimate link between the innate immune response to infection and regulation of the sterol metabolic network characterized by down-regulation of sterol biosynthesis by an interferon regulatory loop mechanism. In time-series experiments profiling genome-wide lipid-associated gene expression of macrophages, we show a selective and coordinated negative regulation of the complete sterol pathway upon viral infection or cytokine treatment with IFNγ or β but not TNF, IL1β, or IL6. Quantitative analysis at the protein level of selected sterol metabolic enzymes upon infection shows a similar level of suppression. Experimental testing of sterol metabolite levels using lipidomic-based measurements shows a reduction in metabolic output. On the basis of pharmacologic and RNAi inhibition of the sterol pathway we show augmented protection against viral infection, and in combination with metabolite rescue experiments, we identify the requirement of the mevalonate-isoprenoid branch of the sterol metabolic network in the protective response upon statin or IFNβ treatment. Conditioned media experiments from infected cells support an involvement of secreted type 1 interferon(s) to be sufficient for reducing the sterol pathway upon infection. Moreover, we show that infection of primary macrophages containing a genetic knockout of the major type I interferon, IFNβ, leads to only a partial suppression of the sterol pathway, while genetic knockout of the receptor for all type I interferon family members, ifnar1, or associated signaling component, tyk2, completely abolishes the reduction of the sterol biosynthetic activity upon infection. Levels of the proteolytically cleaved nuclear forms of SREBP2, a key transcriptional regulator of sterol biosynthesis, are reduced upon infection and IFNβ treatment at both the protein and de novo transcription level. The reduction in srebf2 gene transcription upon infection and IFN treatment is also found to be strictly dependent on ifnar1. Altogether these results show that type 1 IFN signaling is both necessary and sufficient for reducing the sterol metabolic network activity upon infection, thereby linking the regulation of the sterol pathway with interferon anti-viral defense responses. These findings bring a new link between sterol metabolism and interferon antiviral response and support the idea of using host metabolic modifiers of innate immunity as a potential antiviral strategy.

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Effect of a coordinated reduction in multiple enzymes on sterol biosynthesis.(A) Comparison by Western blot analysis of HMGCS1, HMGCR, and SQLE protein levels in mCMV infected (24 hpi) or mock-treated BMDM. Infection was measured by detection of the IE1 mCMV antigen. Intensity values relative to β-actin calculated by densitometry show a decrease of the total amount of protein in the mCMV-infected BMDM compared to the mock-treated samples of 64% for HMGCS1, 50% for HMGCR, and 85% for SQLE. Graphs are representative of two independent experiments with biological duplicates and triplicates, respectively. (B–C) Free cholesterol concentration was determined experimentally by enzymatic assay (Materials and Methods) at 0, 6, 24, 48, and 72 hpi in BMDM (B) and NIH/3T3 cells (C). Cholesterol content is presented as the percentage of free intracellular cholesterol concentration from infected cells compared to mock treatment. Graphs represent the means ± SD of three independent experiments with biological quadruplicates for each experiment. (D) Free cholesterol concentration in BMDM cultures treated with varying concentrations of IFNγ, IFNβ, TNF, IL1β, or IL6. The cholesterol concentration was measured as mentioned above after 48 h post-cytokine treatment. Bars represent means ± SD of two independent experiments with biological quadruplicates for each experiment. *p<0.05, **p<0.01, ***p<0.001, determined with an unpaired Student's t test.
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pbio-1000598-g002: Effect of a coordinated reduction in multiple enzymes on sterol biosynthesis.(A) Comparison by Western blot analysis of HMGCS1, HMGCR, and SQLE protein levels in mCMV infected (24 hpi) or mock-treated BMDM. Infection was measured by detection of the IE1 mCMV antigen. Intensity values relative to β-actin calculated by densitometry show a decrease of the total amount of protein in the mCMV-infected BMDM compared to the mock-treated samples of 64% for HMGCS1, 50% for HMGCR, and 85% for SQLE. Graphs are representative of two independent experiments with biological duplicates and triplicates, respectively. (B–C) Free cholesterol concentration was determined experimentally by enzymatic assay (Materials and Methods) at 0, 6, 24, 48, and 72 hpi in BMDM (B) and NIH/3T3 cells (C). Cholesterol content is presented as the percentage of free intracellular cholesterol concentration from infected cells compared to mock treatment. Graphs represent the means ± SD of three independent experiments with biological quadruplicates for each experiment. (D) Free cholesterol concentration in BMDM cultures treated with varying concentrations of IFNγ, IFNβ, TNF, IL1β, or IL6. The cholesterol concentration was measured as mentioned above after 48 h post-cytokine treatment. Bars represent means ± SD of two independent experiments with biological quadruplicates for each experiment. *p<0.05, **p<0.01, ***p<0.001, determined with an unpaired Student's t test.

Mentions: To independently validate the microarray data described above, Q-RT-PCR analyses of five independent experiments were performed for both infection and IFNγ treatment. In agreement, we find that Q-RT-PCR analysis of selected members of the pathway—Hmgcs1, Hmgcr, IdI1, and Sqle—shows a statistically significant but quantitatively modest reduction in expression (Figure 1C and 1D). Notably, a similar quantitative decrease is also exhibited at the protein level for HMGCS1, HMGCR, and SQLE (Figure 2A).


Host defense against viral infection involves interferon mediated down-regulation of sterol biosynthesis.

Blanc M, Hsieh WY, Robertson KA, Watterson S, Shui G, Lacaze P, Khondoker M, Dickinson P, Sing G, Rodríguez-Martín S, Phelan P, Forster T, Strobl B, Müller M, Riemersma R, Osborne T, Wenk MR, Angulo A, Ghazal P - PLoS Biol. (2011)

Effect of a coordinated reduction in multiple enzymes on sterol biosynthesis.(A) Comparison by Western blot analysis of HMGCS1, HMGCR, and SQLE protein levels in mCMV infected (24 hpi) or mock-treated BMDM. Infection was measured by detection of the IE1 mCMV antigen. Intensity values relative to β-actin calculated by densitometry show a decrease of the total amount of protein in the mCMV-infected BMDM compared to the mock-treated samples of 64% for HMGCS1, 50% for HMGCR, and 85% for SQLE. Graphs are representative of two independent experiments with biological duplicates and triplicates, respectively. (B–C) Free cholesterol concentration was determined experimentally by enzymatic assay (Materials and Methods) at 0, 6, 24, 48, and 72 hpi in BMDM (B) and NIH/3T3 cells (C). Cholesterol content is presented as the percentage of free intracellular cholesterol concentration from infected cells compared to mock treatment. Graphs represent the means ± SD of three independent experiments with biological quadruplicates for each experiment. (D) Free cholesterol concentration in BMDM cultures treated with varying concentrations of IFNγ, IFNβ, TNF, IL1β, or IL6. The cholesterol concentration was measured as mentioned above after 48 h post-cytokine treatment. Bars represent means ± SD of two independent experiments with biological quadruplicates for each experiment. *p<0.05, **p<0.01, ***p<0.001, determined with an unpaired Student's t test.
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pbio-1000598-g002: Effect of a coordinated reduction in multiple enzymes on sterol biosynthesis.(A) Comparison by Western blot analysis of HMGCS1, HMGCR, and SQLE protein levels in mCMV infected (24 hpi) or mock-treated BMDM. Infection was measured by detection of the IE1 mCMV antigen. Intensity values relative to β-actin calculated by densitometry show a decrease of the total amount of protein in the mCMV-infected BMDM compared to the mock-treated samples of 64% for HMGCS1, 50% for HMGCR, and 85% for SQLE. Graphs are representative of two independent experiments with biological duplicates and triplicates, respectively. (B–C) Free cholesterol concentration was determined experimentally by enzymatic assay (Materials and Methods) at 0, 6, 24, 48, and 72 hpi in BMDM (B) and NIH/3T3 cells (C). Cholesterol content is presented as the percentage of free intracellular cholesterol concentration from infected cells compared to mock treatment. Graphs represent the means ± SD of three independent experiments with biological quadruplicates for each experiment. (D) Free cholesterol concentration in BMDM cultures treated with varying concentrations of IFNγ, IFNβ, TNF, IL1β, or IL6. The cholesterol concentration was measured as mentioned above after 48 h post-cytokine treatment. Bars represent means ± SD of two independent experiments with biological quadruplicates for each experiment. *p<0.05, **p<0.01, ***p<0.001, determined with an unpaired Student's t test.
Mentions: To independently validate the microarray data described above, Q-RT-PCR analyses of five independent experiments were performed for both infection and IFNγ treatment. In agreement, we find that Q-RT-PCR analysis of selected members of the pathway—Hmgcs1, Hmgcr, IdI1, and Sqle—shows a statistically significant but quantitatively modest reduction in expression (Figure 1C and 1D). Notably, a similar quantitative decrease is also exhibited at the protein level for HMGCS1, HMGCR, and SQLE (Figure 2A).

Bottom Line: Experimental testing of sterol metabolite levels using lipidomic-based measurements shows a reduction in metabolic output.Altogether these results show that type 1 IFN signaling is both necessary and sufficient for reducing the sterol metabolic network activity upon infection, thereby linking the regulation of the sterol pathway with interferon anti-viral defense responses.These findings bring a new link between sterol metabolism and interferon antiviral response and support the idea of using host metabolic modifiers of innate immunity as a potential antiviral strategy.

View Article: PubMed Central - PubMed

Affiliation: Division of Pathway Medicine and Centre for Infectious Diseases, University of Edinburgh, Edinburgh, United Kingdom.

ABSTRACT
Little is known about the protective role of inflammatory processes in modulating lipid metabolism in infection. Here we report an intimate link between the innate immune response to infection and regulation of the sterol metabolic network characterized by down-regulation of sterol biosynthesis by an interferon regulatory loop mechanism. In time-series experiments profiling genome-wide lipid-associated gene expression of macrophages, we show a selective and coordinated negative regulation of the complete sterol pathway upon viral infection or cytokine treatment with IFNγ or β but not TNF, IL1β, or IL6. Quantitative analysis at the protein level of selected sterol metabolic enzymes upon infection shows a similar level of suppression. Experimental testing of sterol metabolite levels using lipidomic-based measurements shows a reduction in metabolic output. On the basis of pharmacologic and RNAi inhibition of the sterol pathway we show augmented protection against viral infection, and in combination with metabolite rescue experiments, we identify the requirement of the mevalonate-isoprenoid branch of the sterol metabolic network in the protective response upon statin or IFNβ treatment. Conditioned media experiments from infected cells support an involvement of secreted type 1 interferon(s) to be sufficient for reducing the sterol pathway upon infection. Moreover, we show that infection of primary macrophages containing a genetic knockout of the major type I interferon, IFNβ, leads to only a partial suppression of the sterol pathway, while genetic knockout of the receptor for all type I interferon family members, ifnar1, or associated signaling component, tyk2, completely abolishes the reduction of the sterol biosynthetic activity upon infection. Levels of the proteolytically cleaved nuclear forms of SREBP2, a key transcriptional regulator of sterol biosynthesis, are reduced upon infection and IFNβ treatment at both the protein and de novo transcription level. The reduction in srebf2 gene transcription upon infection and IFN treatment is also found to be strictly dependent on ifnar1. Altogether these results show that type 1 IFN signaling is both necessary and sufficient for reducing the sterol metabolic network activity upon infection, thereby linking the regulation of the sterol pathway with interferon anti-viral defense responses. These findings bring a new link between sterol metabolism and interferon antiviral response and support the idea of using host metabolic modifiers of innate immunity as a potential antiviral strategy.

Show MeSH
Related in: MedlinePlus