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Allele-specific induction of IL-1β expression by C/EBPβ and PU.1 contributes to increased tuberculosis susceptibility.

Zhang G, Zhou B, Li S, Yue J, Yang H, Wen Y, Zhan S, Wang W, Liao M, Zhang M, Zeng G, Feng CG, Sassetti CM, Chen X - PLoS Pathog. (2014)

Bottom Line: Mycobacterium tuberculosis infection is associated with a spectrum of clinical outcomes, from long-term latent infection to different manifestations of progressive disease.Thus, the ultimate contribution of individual inflammatory pathways to the outcome of M. tuberculosis infection remains ambiguous.In this study, we identified a naturally-occurring polymorphism in the human IL1B promoter region, which alters the association of the C/EBPβ and PU.1 transcription factors and controls Mtb-induced IL-1β production.

View Article: PubMed Central - PubMed

Affiliation: Guangdong Key Lab of Emerging Infectious Diseases, Guangdong Medical College, Shenzhen, China; Shenzhen Key Lab of Infection and Immunity, Shenzhen Third People's Hospital, Guangdong Medical College, Shenzhen, China.

ABSTRACT
Mycobacterium tuberculosis infection is associated with a spectrum of clinical outcomes, from long-term latent infection to different manifestations of progressive disease. Pro-inflammatory pathways, such as those controlled by IL-1β, have the contrasting potential both to prevent disease by restricting bacterial replication, and to promote disease by inflicting tissue damage. Thus, the ultimate contribution of individual inflammatory pathways to the outcome of M. tuberculosis infection remains ambiguous. In this study, we identified a naturally-occurring polymorphism in the human IL1B promoter region, which alters the association of the C/EBPβ and PU.1 transcription factors and controls Mtb-induced IL-1β production. The high-IL-1β expressing genotype was associated with the development of active tuberculosis, the severity of pulmonary disease and poor treatment outcome in TB patients. Higher IL-1β expression did not suppress the activity of IFN-γ-producing T cells, but instead correlated with neutrophil accumulation in the lung. These observations support a specific role for IL-1β and granulocytic inflammation as a driver of TB disease progression in humans, and suggest novel strategies for the prevention and treatment of tuberculosis.

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SNP rs1143627 variant affects C/EBPβ and PU.1 binding to the IL1B promoter.(A and B) Nuclear extracts were prepared PMA-differentiated U937 cells stimulated without (A) or with (B) Mtb lysate (20 µg/mL) for 24 h. Nuclear extracts were incubated with T-probe (lanes 1–4) or C-probe (lanes 5–8) in the absence or presence of unlabelled competitors and subjected to EMSA. (C) The relative abundance of complex 1 bound to T probe or C probe in (B) quantified by densitometry. Data are shown as the mean ± SEM, and differences between groups were compared with unpaired t tests. (D) Nuclear extracts prepared from (A) were incubated with T-probe in the absence (lane 2) or presence of antibodies against transcription factors including ZEB1, MEF2, C/EBPα, C/EBPβ, PU.1, TBP, and SP1 (lanes 4–10). Anti-C/EBPβ (lane 7) and anti-PU.1 (lane 8) supershift the DNA-protein complex 1. *, p<0.05.
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ppat-1004426-g004: SNP rs1143627 variant affects C/EBPβ and PU.1 binding to the IL1B promoter.(A and B) Nuclear extracts were prepared PMA-differentiated U937 cells stimulated without (A) or with (B) Mtb lysate (20 µg/mL) for 24 h. Nuclear extracts were incubated with T-probe (lanes 1–4) or C-probe (lanes 5–8) in the absence or presence of unlabelled competitors and subjected to EMSA. (C) The relative abundance of complex 1 bound to T probe or C probe in (B) quantified by densitometry. Data are shown as the mean ± SEM, and differences between groups were compared with unpaired t tests. (D) Nuclear extracts prepared from (A) were incubated with T-probe in the absence (lane 2) or presence of antibodies against transcription factors including ZEB1, MEF2, C/EBPα, C/EBPβ, PU.1, TBP, and SP1 (lanes 4–10). Anti-C/EBPβ (lane 7) and anti-PU.1 (lane 8) supershift the DNA-protein complex 1. *, p<0.05.

Mentions: The rs1143627 defines a T>C mutation at the -31 position of the IL1Β promoter. We used electrophoretic mobility-shift analysis (EMSA) to determine if this polymorphism altered protein binding to this promoter region. Synthetic allele-specific oligonucleotides representing the polymorphic rs1143627 sites were incubated with nuclear protein extracts from human monocytic U937 cells stimulated without or with killed Mtb lysate. There was no difference in binding activity between rs1143627T oligonucleotide (T Probe) and rs1143627C oligonucleotide (C Probe) when U937 were not stimulated with Mtb lysate (Fig. 4A). Previous Mtb stimulation of these cells induced the formation of a DNA binding complex on the rs1143627T oligonucleotide (“complex 1”, Figure 4B). In contrast, Mtb exposure induced less complex 1 formation on the rs1143627C oligonucleotide (Fig. 4B, lane 2 and 6; Fig. 4C). Furthermore, complex 1 formation on radiolabelled rs1143627 was specifically blocked by competition with the unlabelled oligonucleotide (Fig. 4B, lanes 3, 4, 7, and 8). These results indicated that the binding of one or more proteins to the IL1B promoter region was altered by rs1143627 polymorphism, which could cause the observed difference in IL-1β expression that was associated with these genotypes.


Allele-specific induction of IL-1β expression by C/EBPβ and PU.1 contributes to increased tuberculosis susceptibility.

Zhang G, Zhou B, Li S, Yue J, Yang H, Wen Y, Zhan S, Wang W, Liao M, Zhang M, Zeng G, Feng CG, Sassetti CM, Chen X - PLoS Pathog. (2014)

SNP rs1143627 variant affects C/EBPβ and PU.1 binding to the IL1B promoter.(A and B) Nuclear extracts were prepared PMA-differentiated U937 cells stimulated without (A) or with (B) Mtb lysate (20 µg/mL) for 24 h. Nuclear extracts were incubated with T-probe (lanes 1–4) or C-probe (lanes 5–8) in the absence or presence of unlabelled competitors and subjected to EMSA. (C) The relative abundance of complex 1 bound to T probe or C probe in (B) quantified by densitometry. Data are shown as the mean ± SEM, and differences between groups were compared with unpaired t tests. (D) Nuclear extracts prepared from (A) were incubated with T-probe in the absence (lane 2) or presence of antibodies against transcription factors including ZEB1, MEF2, C/EBPα, C/EBPβ, PU.1, TBP, and SP1 (lanes 4–10). Anti-C/EBPβ (lane 7) and anti-PU.1 (lane 8) supershift the DNA-protein complex 1. *, p<0.05.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4199770&req=5

ppat-1004426-g004: SNP rs1143627 variant affects C/EBPβ and PU.1 binding to the IL1B promoter.(A and B) Nuclear extracts were prepared PMA-differentiated U937 cells stimulated without (A) or with (B) Mtb lysate (20 µg/mL) for 24 h. Nuclear extracts were incubated with T-probe (lanes 1–4) or C-probe (lanes 5–8) in the absence or presence of unlabelled competitors and subjected to EMSA. (C) The relative abundance of complex 1 bound to T probe or C probe in (B) quantified by densitometry. Data are shown as the mean ± SEM, and differences between groups were compared with unpaired t tests. (D) Nuclear extracts prepared from (A) were incubated with T-probe in the absence (lane 2) or presence of antibodies against transcription factors including ZEB1, MEF2, C/EBPα, C/EBPβ, PU.1, TBP, and SP1 (lanes 4–10). Anti-C/EBPβ (lane 7) and anti-PU.1 (lane 8) supershift the DNA-protein complex 1. *, p<0.05.
Mentions: The rs1143627 defines a T>C mutation at the -31 position of the IL1Β promoter. We used electrophoretic mobility-shift analysis (EMSA) to determine if this polymorphism altered protein binding to this promoter region. Synthetic allele-specific oligonucleotides representing the polymorphic rs1143627 sites were incubated with nuclear protein extracts from human monocytic U937 cells stimulated without or with killed Mtb lysate. There was no difference in binding activity between rs1143627T oligonucleotide (T Probe) and rs1143627C oligonucleotide (C Probe) when U937 were not stimulated with Mtb lysate (Fig. 4A). Previous Mtb stimulation of these cells induced the formation of a DNA binding complex on the rs1143627T oligonucleotide (“complex 1”, Figure 4B). In contrast, Mtb exposure induced less complex 1 formation on the rs1143627C oligonucleotide (Fig. 4B, lane 2 and 6; Fig. 4C). Furthermore, complex 1 formation on radiolabelled rs1143627 was specifically blocked by competition with the unlabelled oligonucleotide (Fig. 4B, lanes 3, 4, 7, and 8). These results indicated that the binding of one or more proteins to the IL1B promoter region was altered by rs1143627 polymorphism, which could cause the observed difference in IL-1β expression that was associated with these genotypes.

Bottom Line: Mycobacterium tuberculosis infection is associated with a spectrum of clinical outcomes, from long-term latent infection to different manifestations of progressive disease.Thus, the ultimate contribution of individual inflammatory pathways to the outcome of M. tuberculosis infection remains ambiguous.In this study, we identified a naturally-occurring polymorphism in the human IL1B promoter region, which alters the association of the C/EBPβ and PU.1 transcription factors and controls Mtb-induced IL-1β production.

View Article: PubMed Central - PubMed

Affiliation: Guangdong Key Lab of Emerging Infectious Diseases, Guangdong Medical College, Shenzhen, China; Shenzhen Key Lab of Infection and Immunity, Shenzhen Third People's Hospital, Guangdong Medical College, Shenzhen, China.

ABSTRACT
Mycobacterium tuberculosis infection is associated with a spectrum of clinical outcomes, from long-term latent infection to different manifestations of progressive disease. Pro-inflammatory pathways, such as those controlled by IL-1β, have the contrasting potential both to prevent disease by restricting bacterial replication, and to promote disease by inflicting tissue damage. Thus, the ultimate contribution of individual inflammatory pathways to the outcome of M. tuberculosis infection remains ambiguous. In this study, we identified a naturally-occurring polymorphism in the human IL1B promoter region, which alters the association of the C/EBPβ and PU.1 transcription factors and controls Mtb-induced IL-1β production. The high-IL-1β expressing genotype was associated with the development of active tuberculosis, the severity of pulmonary disease and poor treatment outcome in TB patients. Higher IL-1β expression did not suppress the activity of IFN-γ-producing T cells, but instead correlated with neutrophil accumulation in the lung. These observations support a specific role for IL-1β and granulocytic inflammation as a driver of TB disease progression in humans, and suggest novel strategies for the prevention and treatment of tuberculosis.

Show MeSH
Related in: MedlinePlus