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Vitamin D induces interleukin-1β expression: paracrine macrophage epithelial signaling controls M. tuberculosis infection.

Verway M, Bouttier M, Wang TT, Carrier M, Calderon M, An BS, Devemy E, McIntosh F, Divangahi M, Behr MA, White JH - PLoS Pathog. (2013)

Bottom Line: We found that the virulent Mtb strain H37Rv elicits a broad host transcriptional response.Secretion of IL-1β from infected cells required the NLRP3/caspase-1 inflammasome.Co-culture significantly prolonged survival of infected macrophages, and 1,25D/infection-induced IL-1β secretion from macrophages reduced mycobacterial burden by stimulating the anti-mycobacterial capacity of co-cultured lung epithelial cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, McGill University, Montreal, Quebec, Canada.

ABSTRACT
Although vitamin D deficiency is a common feature among patients presenting with active tuberculosis, the full scope of vitamin D action during Mycobacterium tuberculosis (Mtb) infection is poorly understood. As macrophages are the primary site of Mtb infection and are sites of vitamin D signaling, we have used these cells to understand the molecular mechanisms underlying modulation of the immune response by the hormonal form of vitamin D, 1,25-dihydroxyvitamin D (1,25D). We found that the virulent Mtb strain H37Rv elicits a broad host transcriptional response. Transcriptome profiling also revealed that the profile of target genes regulated by 1,25D is substantially altered by infection, and that 1,25D generally boosts infection-stimulated cytokine/chemokine responses. We further focused on the role of 1,25D- and infection-induced interleukin 1β (IL-1β) expression in response to infection. 1,25D enhanced IL-1β expression via a direct transcriptional mechanism. Secretion of IL-1β from infected cells required the NLRP3/caspase-1 inflammasome. The impact of IL-1β production was investigated in a novel model wherein infected macrophages were co-cultured with primary human small airway epithelial cells. Co-culture significantly prolonged survival of infected macrophages, and 1,25D/infection-induced IL-1β secretion from macrophages reduced mycobacterial burden by stimulating the anti-mycobacterial capacity of co-cultured lung epithelial cells. These effects were independent of 1,25D-stimulated autophagy in macrophages but dependent upon epithelial IL1R1 signaling and IL-1β-driven epithelial production of the antimicrobial peptide DEFB4/HBD2. These data provide evidence that the anti-microbial actions of vitamin D extend beyond the macrophage by modulating paracrine signaling, reinforcing its role in innate immune regulation in humans.

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1,25D directly regulates IL-1β expression in Mtb-infected macrophages.(A) Expression of IL1B transcripts as assessed by RT/qPCR in control or H37Rv-infected THP-1 cells 24 hours after infection. Data are normalized to the uninfected, untreated control (NI, uninfected cells; NI+D, uninfected cells treated with 1,25D; I, H37Rv-infected cells; I+D, infected cells treated with 1,25D). (B) Expression of IL1B transcripts in two independent cultures of H37Rv-infected primary human macrophages, analyzed as in A. (C) Protein expression and processing of IL-1β in uninfected and infected THP-1 cells. (D) Secretion of IL-1β from uninfected and infected THP-1 cells. (E) Secretion of IL-1β from uninfected and infected primary human macrophages. (F) ChIP analysis of association of the VDR with the IL1B VDRE and transcription start site (TSS). (G) ChIP analysis of recruitment of the large subunit of RNA PolII to the VDRE and TSS of the IL1B gene. ChIP values are normalized to input for each condition and expressed as a fold relative to non-specific IgG control. All data are from one experiment and representative of at least three independent experiments (n = 3, mean, s.d.). *P<0.05, **P<0.01, ***P<0.001 as determined by Student's t-test.
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ppat-1003407-g002: 1,25D directly regulates IL-1β expression in Mtb-infected macrophages.(A) Expression of IL1B transcripts as assessed by RT/qPCR in control or H37Rv-infected THP-1 cells 24 hours after infection. Data are normalized to the uninfected, untreated control (NI, uninfected cells; NI+D, uninfected cells treated with 1,25D; I, H37Rv-infected cells; I+D, infected cells treated with 1,25D). (B) Expression of IL1B transcripts in two independent cultures of H37Rv-infected primary human macrophages, analyzed as in A. (C) Protein expression and processing of IL-1β in uninfected and infected THP-1 cells. (D) Secretion of IL-1β from uninfected and infected THP-1 cells. (E) Secretion of IL-1β from uninfected and infected primary human macrophages. (F) ChIP analysis of association of the VDR with the IL1B VDRE and transcription start site (TSS). (G) ChIP analysis of recruitment of the large subunit of RNA PolII to the VDRE and TSS of the IL1B gene. ChIP values are normalized to input for each condition and expressed as a fold relative to non-specific IgG control. All data are from one experiment and representative of at least three independent experiments (n = 3, mean, s.d.). *P<0.05, **P<0.01, ***P<0.001 as determined by Student's t-test.

Mentions: Considering the critical role of IL-1β in immunity to Mtb infection [8], [9], [41], we next investigated the mechanisms by which 1,25D increased the expression and secretion of IL-1β in Mtb-infected macrophages. Consistent with the microarray data, RT/qPCR analysis showed that 1,25D up-regulated the expression of IL1B transcripts in uninfected and Mtb-infected macrophages (Figure 2A). Essentially identical results were obtained with two independent cultures of primary human macrophages (Figure 2B). Although the levels of pro-IL-1β were elevated in both 1,25D-treated macrophages as well as Mtb-infected macrophages treated with 1,25D, mature IL-1β was only detected in extracts of Mtb-infected cells treated with 1,25D (Figure 2C). Results of western blotting were consistent with analysis of IL-1β released from THP-1 cells or primary human macrophages, where secretion was observed only in supernatants of infected cells, and significantly elevated upon treatment with 1,25D (Figures 2D, E).


Vitamin D induces interleukin-1β expression: paracrine macrophage epithelial signaling controls M. tuberculosis infection.

Verway M, Bouttier M, Wang TT, Carrier M, Calderon M, An BS, Devemy E, McIntosh F, Divangahi M, Behr MA, White JH - PLoS Pathog. (2013)

1,25D directly regulates IL-1β expression in Mtb-infected macrophages.(A) Expression of IL1B transcripts as assessed by RT/qPCR in control or H37Rv-infected THP-1 cells 24 hours after infection. Data are normalized to the uninfected, untreated control (NI, uninfected cells; NI+D, uninfected cells treated with 1,25D; I, H37Rv-infected cells; I+D, infected cells treated with 1,25D). (B) Expression of IL1B transcripts in two independent cultures of H37Rv-infected primary human macrophages, analyzed as in A. (C) Protein expression and processing of IL-1β in uninfected and infected THP-1 cells. (D) Secretion of IL-1β from uninfected and infected THP-1 cells. (E) Secretion of IL-1β from uninfected and infected primary human macrophages. (F) ChIP analysis of association of the VDR with the IL1B VDRE and transcription start site (TSS). (G) ChIP analysis of recruitment of the large subunit of RNA PolII to the VDRE and TSS of the IL1B gene. ChIP values are normalized to input for each condition and expressed as a fold relative to non-specific IgG control. All data are from one experiment and representative of at least three independent experiments (n = 3, mean, s.d.). *P<0.05, **P<0.01, ***P<0.001 as determined by Student's t-test.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675149&req=5

ppat-1003407-g002: 1,25D directly regulates IL-1β expression in Mtb-infected macrophages.(A) Expression of IL1B transcripts as assessed by RT/qPCR in control or H37Rv-infected THP-1 cells 24 hours after infection. Data are normalized to the uninfected, untreated control (NI, uninfected cells; NI+D, uninfected cells treated with 1,25D; I, H37Rv-infected cells; I+D, infected cells treated with 1,25D). (B) Expression of IL1B transcripts in two independent cultures of H37Rv-infected primary human macrophages, analyzed as in A. (C) Protein expression and processing of IL-1β in uninfected and infected THP-1 cells. (D) Secretion of IL-1β from uninfected and infected THP-1 cells. (E) Secretion of IL-1β from uninfected and infected primary human macrophages. (F) ChIP analysis of association of the VDR with the IL1B VDRE and transcription start site (TSS). (G) ChIP analysis of recruitment of the large subunit of RNA PolII to the VDRE and TSS of the IL1B gene. ChIP values are normalized to input for each condition and expressed as a fold relative to non-specific IgG control. All data are from one experiment and representative of at least three independent experiments (n = 3, mean, s.d.). *P<0.05, **P<0.01, ***P<0.001 as determined by Student's t-test.
Mentions: Considering the critical role of IL-1β in immunity to Mtb infection [8], [9], [41], we next investigated the mechanisms by which 1,25D increased the expression and secretion of IL-1β in Mtb-infected macrophages. Consistent with the microarray data, RT/qPCR analysis showed that 1,25D up-regulated the expression of IL1B transcripts in uninfected and Mtb-infected macrophages (Figure 2A). Essentially identical results were obtained with two independent cultures of primary human macrophages (Figure 2B). Although the levels of pro-IL-1β were elevated in both 1,25D-treated macrophages as well as Mtb-infected macrophages treated with 1,25D, mature IL-1β was only detected in extracts of Mtb-infected cells treated with 1,25D (Figure 2C). Results of western blotting were consistent with analysis of IL-1β released from THP-1 cells or primary human macrophages, where secretion was observed only in supernatants of infected cells, and significantly elevated upon treatment with 1,25D (Figures 2D, E).

Bottom Line: We found that the virulent Mtb strain H37Rv elicits a broad host transcriptional response.Secretion of IL-1β from infected cells required the NLRP3/caspase-1 inflammasome.Co-culture significantly prolonged survival of infected macrophages, and 1,25D/infection-induced IL-1β secretion from macrophages reduced mycobacterial burden by stimulating the anti-mycobacterial capacity of co-cultured lung epithelial cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, McGill University, Montreal, Quebec, Canada.

ABSTRACT
Although vitamin D deficiency is a common feature among patients presenting with active tuberculosis, the full scope of vitamin D action during Mycobacterium tuberculosis (Mtb) infection is poorly understood. As macrophages are the primary site of Mtb infection and are sites of vitamin D signaling, we have used these cells to understand the molecular mechanisms underlying modulation of the immune response by the hormonal form of vitamin D, 1,25-dihydroxyvitamin D (1,25D). We found that the virulent Mtb strain H37Rv elicits a broad host transcriptional response. Transcriptome profiling also revealed that the profile of target genes regulated by 1,25D is substantially altered by infection, and that 1,25D generally boosts infection-stimulated cytokine/chemokine responses. We further focused on the role of 1,25D- and infection-induced interleukin 1β (IL-1β) expression in response to infection. 1,25D enhanced IL-1β expression via a direct transcriptional mechanism. Secretion of IL-1β from infected cells required the NLRP3/caspase-1 inflammasome. The impact of IL-1β production was investigated in a novel model wherein infected macrophages were co-cultured with primary human small airway epithelial cells. Co-culture significantly prolonged survival of infected macrophages, and 1,25D/infection-induced IL-1β secretion from macrophages reduced mycobacterial burden by stimulating the anti-mycobacterial capacity of co-cultured lung epithelial cells. These effects were independent of 1,25D-stimulated autophagy in macrophages but dependent upon epithelial IL1R1 signaling and IL-1β-driven epithelial production of the antimicrobial peptide DEFB4/HBD2. These data provide evidence that the anti-microbial actions of vitamin D extend beyond the macrophage by modulating paracrine signaling, reinforcing its role in innate immune regulation in humans.

Show MeSH
Related in: MedlinePlus