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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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Related in: MedlinePlus

Regulation of the cholesterol pathway upon mCMV infection.(A) The Sterol biosynthesis pathway shown in KEGG notation with abbreviated metabolites (abbreviations listed in Text S1). The geranylgeranylation pathway responsible for GGPP synthesis is shown in the dashed box. (B) Heat map of the cholesterol biosynthesis temporal genes' expression during the first 12 h of mCMV infection (left panel) or IFNγ treatment (right panel). Each time point corresponds to one independent biological sample, and columns indicate time in hours. Fold changes of expression levels are represented on a Log2 scale compared to mock-treated cells, ranging from a 0.8× lower expression (dark blue) to a 1.2× higher expression (bright yellow). (C–H) Expression analysis measured by qRT-PCR of Hmgcs1, Hmgcr, Idi1, and Sqle genes in BMDM infected with mCMV(24 hpi) (C) or treated for 24 h with IFNγ (10 and 100 U/ml) (D), IFNβ (10 and 25 U/ml) (E), IL6 (10 and 25 U/ml) (F), IL1β (10 and 100 U/ml) (G), or TNF (10 and 100 U/ml) (H). Graphs show levels of mRNA expression of the respective genes either infected or cytokines-treated relative to mock samples. Bars represent the means ± SD of five independent experiments with biological triplicates 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-g001: Regulation of the cholesterol pathway upon mCMV infection.(A) The Sterol biosynthesis pathway shown in KEGG notation with abbreviated metabolites (abbreviations listed in Text S1). The geranylgeranylation pathway responsible for GGPP synthesis is shown in the dashed box. (B) Heat map of the cholesterol biosynthesis temporal genes' expression during the first 12 h of mCMV infection (left panel) or IFNγ treatment (right panel). Each time point corresponds to one independent biological sample, and columns indicate time in hours. Fold changes of expression levels are represented on a Log2 scale compared to mock-treated cells, ranging from a 0.8× lower expression (dark blue) to a 1.2× higher expression (bright yellow). (C–H) Expression analysis measured by qRT-PCR of Hmgcs1, Hmgcr, Idi1, and Sqle genes in BMDM infected with mCMV(24 hpi) (C) or treated for 24 h with IFNγ (10 and 100 U/ml) (D), IFNβ (10 and 25 U/ml) (E), IL6 (10 and 25 U/ml) (F), IL1β (10 and 100 U/ml) (G), or TNF (10 and 100 U/ml) (H). Graphs show levels of mRNA expression of the respective genes either infected or cytokines-treated relative to mock samples. Bars represent the means ± SD of five independent experiments with biological triplicates for each experiment. *p<0.05, **p<0.01, ***p<0.001, determined with an unpaired Student's t test.

Mentions: As a first step, an integrative approach combining bioinformatics tools and a time-series analysis of gene expression changes was applied to mCMV-infected or interferon (IFN) γ-activated primary bone-marrow-derived macrophages (BMDM). These primary BMDM cultures represent a physiologically relevant cell system for the combined analysis of infection, inflammation, and lipogenesis [44]–[46]. In the following experiments, infected or IFNγ-treated BMDM RNA was harvested every 30 min up to 12 h post-challenge for microarray gene expression profiling. In this study, analysis of expression data was exclusively restricted to lipogenic-associated genes. For this purpose, a combination of literature and data-mining identified over one thousand genes with published direct or indirect functions relating to cellular lipid metabolism, regulation, and synthesis (Text S1). When this resource was used to interrogate a subset of our time-series data which passed a stringent filtering threshold (p<10−6), 89% of lipogenic-associated genes were detected, of which 12% were significantly regulated (113/958) upon IFNγ treatment and 23% were significantly altered in their expression (195/958) after mCMV infection. This represented a significant and highly selective lipogenic response (Figure S1) with altered genes showing a high degree of overlap between infection and IFNγ activation (Table S1). Notably, clear differences in the specific class of lipogenic genes in up- and down-regulated groups were observed. Of the IFNγ down-regulated transcripts, a significant proportion (14/35, 40%) were related to the sterol pathway, while fatty acid pathways were pre-eminent (6/35, 17%) in the up-regulated gene group (Figure S1C). A statistical evaluation investigating pathway over-representation indicated a highly pathway-specific response including previously known pathways for inositol (Table S2E–F) [47] perturbed by mCMV infection. Significantly, however, the most pronounced pathway changes in the down-regulated genes common to both stimuli were associated with sterol lipid metabolism (Table S2E and Figure 1A), which exhibited a gradual, temporal decline in expression from 6 h post-infection (hpi) onwards (Figure 1B). Additional microarray experiments to further explore this observation revealed a further reduction in sterol pathway gene expression observed at 24 hpi (unpublished data). It is worth noting, however, that the observed level of reduction in expression for any particular transcript was relatively modest (ranging from 1.3- to 5-fold for infection and 1.3- to 3-fold for IFNγ treatment over a 24 h time frame).


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)

Regulation of the cholesterol pathway upon mCMV infection.(A) The Sterol biosynthesis pathway shown in KEGG notation with abbreviated metabolites (abbreviations listed in Text S1). The geranylgeranylation pathway responsible for GGPP synthesis is shown in the dashed box. (B) Heat map of the cholesterol biosynthesis temporal genes' expression during the first 12 h of mCMV infection (left panel) or IFNγ treatment (right panel). Each time point corresponds to one independent biological sample, and columns indicate time in hours. Fold changes of expression levels are represented on a Log2 scale compared to mock-treated cells, ranging from a 0.8× lower expression (dark blue) to a 1.2× higher expression (bright yellow). (C–H) Expression analysis measured by qRT-PCR of Hmgcs1, Hmgcr, Idi1, and Sqle genes in BMDM infected with mCMV(24 hpi) (C) or treated for 24 h with IFNγ (10 and 100 U/ml) (D), IFNβ (10 and 25 U/ml) (E), IL6 (10 and 25 U/ml) (F), IL1β (10 and 100 U/ml) (G), or TNF (10 and 100 U/ml) (H). Graphs show levels of mRNA expression of the respective genes either infected or cytokines-treated relative to mock samples. Bars represent the means ± SD of five independent experiments with biological triplicates for each experiment. *p<0.05, **p<0.01, ***p<0.001, determined with an unpaired Student's t test.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3050939&req=5

pbio-1000598-g001: Regulation of the cholesterol pathway upon mCMV infection.(A) The Sterol biosynthesis pathway shown in KEGG notation with abbreviated metabolites (abbreviations listed in Text S1). The geranylgeranylation pathway responsible for GGPP synthesis is shown in the dashed box. (B) Heat map of the cholesterol biosynthesis temporal genes' expression during the first 12 h of mCMV infection (left panel) or IFNγ treatment (right panel). Each time point corresponds to one independent biological sample, and columns indicate time in hours. Fold changes of expression levels are represented on a Log2 scale compared to mock-treated cells, ranging from a 0.8× lower expression (dark blue) to a 1.2× higher expression (bright yellow). (C–H) Expression analysis measured by qRT-PCR of Hmgcs1, Hmgcr, Idi1, and Sqle genes in BMDM infected with mCMV(24 hpi) (C) or treated for 24 h with IFNγ (10 and 100 U/ml) (D), IFNβ (10 and 25 U/ml) (E), IL6 (10 and 25 U/ml) (F), IL1β (10 and 100 U/ml) (G), or TNF (10 and 100 U/ml) (H). Graphs show levels of mRNA expression of the respective genes either infected or cytokines-treated relative to mock samples. Bars represent the means ± SD of five independent experiments with biological triplicates for each experiment. *p<0.05, **p<0.01, ***p<0.001, determined with an unpaired Student's t test.
Mentions: As a first step, an integrative approach combining bioinformatics tools and a time-series analysis of gene expression changes was applied to mCMV-infected or interferon (IFN) γ-activated primary bone-marrow-derived macrophages (BMDM). These primary BMDM cultures represent a physiologically relevant cell system for the combined analysis of infection, inflammation, and lipogenesis [44]–[46]. In the following experiments, infected or IFNγ-treated BMDM RNA was harvested every 30 min up to 12 h post-challenge for microarray gene expression profiling. In this study, analysis of expression data was exclusively restricted to lipogenic-associated genes. For this purpose, a combination of literature and data-mining identified over one thousand genes with published direct or indirect functions relating to cellular lipid metabolism, regulation, and synthesis (Text S1). When this resource was used to interrogate a subset of our time-series data which passed a stringent filtering threshold (p<10−6), 89% of lipogenic-associated genes were detected, of which 12% were significantly regulated (113/958) upon IFNγ treatment and 23% were significantly altered in their expression (195/958) after mCMV infection. This represented a significant and highly selective lipogenic response (Figure S1) with altered genes showing a high degree of overlap between infection and IFNγ activation (Table S1). Notably, clear differences in the specific class of lipogenic genes in up- and down-regulated groups were observed. Of the IFNγ down-regulated transcripts, a significant proportion (14/35, 40%) were related to the sterol pathway, while fatty acid pathways were pre-eminent (6/35, 17%) in the up-regulated gene group (Figure S1C). A statistical evaluation investigating pathway over-representation indicated a highly pathway-specific response including previously known pathways for inositol (Table S2E–F) [47] perturbed by mCMV infection. Significantly, however, the most pronounced pathway changes in the down-regulated genes common to both stimuli were associated with sterol lipid metabolism (Table S2E and Figure 1A), which exhibited a gradual, temporal decline in expression from 6 h post-infection (hpi) onwards (Figure 1B). Additional microarray experiments to further explore this observation revealed a further reduction in sterol pathway gene expression observed at 24 hpi (unpublished data). It is worth noting, however, that the observed level of reduction in expression for any particular transcript was relatively modest (ranging from 1.3- to 5-fold for infection and 1.3- to 3-fold for IFNγ treatment over a 24 h time frame).

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