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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.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.

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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DEFB4 and CAMP genes are regulated by IL-1β and 1,25D in SAECs.(A) Expression of DEFB4 and CAMP in SAECs as measured by RT/qPCR. Cells were incubated with IL-1β (10 ng/ml) or 1,25D (100 nM) for 24 h. (B) Media supernatants from cells in (A) were tested for DEFB4 and LL-37 protein secretion by ELISA. (C) and (D) Expression of DEFB4 (C) and CAMP (D) genes in SAECs incubated with conditioned media from uninfected (NI) or H37Rv-infected (I) THP-1 cells treated with vehicle or 100 nM 1,25D (+D) for 24 hours. Media was treated with neutralizing antibody against IL-1β (α-IL-1β), or normal serum IgG, as indicated, for 30 minutes prior to incubation with cells. Values are expressed as a fold of the NI control. All data are from one experiment and representative of three independent experiments using separate donors of SAECs (n = 3, mean, s.d.). *P<0.05, **P<0.01 as determined by Student's t-test relative untreated (A, B) or respective IgG (C) control.
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ppat-1003407-g004: DEFB4 and CAMP genes are regulated by IL-1β and 1,25D in SAECs.(A) Expression of DEFB4 and CAMP in SAECs as measured by RT/qPCR. Cells were incubated with IL-1β (10 ng/ml) or 1,25D (100 nM) for 24 h. (B) Media supernatants from cells in (A) were tested for DEFB4 and LL-37 protein secretion by ELISA. (C) and (D) Expression of DEFB4 (C) and CAMP (D) genes in SAECs incubated with conditioned media from uninfected (NI) or H37Rv-infected (I) THP-1 cells treated with vehicle or 100 nM 1,25D (+D) for 24 hours. Media was treated with neutralizing antibody against IL-1β (α-IL-1β), or normal serum IgG, as indicated, for 30 minutes prior to incubation with cells. Values are expressed as a fold of the NI control. All data are from one experiment and representative of three independent experiments using separate donors of SAECs (n = 3, mean, s.d.). *P<0.05, **P<0.01 as determined by Student's t-test relative untreated (A, B) or respective IgG (C) control.

Mentions: Upon infection, alveolar macrophages initially phagocytose Mtb. Importantly, alveolar macrophages are also in direct contact with the respiratory epithelial surface [4]. Although the immunological contribution of lung epithelia is well studied in other contexts [52], it is poorly characterized during the course of Mtb infection. IL-1β induces expression of genes encoding AMPs in epithelial cells through its capacity to stimulate the activity of transcription factor, NF-κB, leading to secretion of antimicrobial proteins [53], [54]. Thus, to understand the potential crosstalk between alveolar macrophages and the human respiratory epithelial surface, we conducted a series of experiments using an in vitro co-culture system between macrophages and human primary cultures of non-polarized small airway epithelial cells (SAECs) from multiple donors. In cultures of SAECs alone, recombinant IL-1β induced expression of the gene encoding DEFB4, while 1,25D had no significant effect (Figure 4A). Conversely, induction of CAMP gene expression was largely 1,25D-dependent (Figure 4A). Media supernatants from these treated SAECs were assayed for DEFB4 and CAMP secretion by ELISA. DEFB4 was detected under control conditions, and was found to be elevated when SAECs were treated with rIL-1β, but not 1,25D (Figure 4B). In contrast, the cleaved C-terminal of CAMP, LL-37, was not detected above background signal under any of the conditions (Figure 4B). In comparison, neither DEFB4 nor LL-37 was detected in media supernatants from THP-1 cells infected with Mtb using this assay (data not shown).


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)

DEFB4 and CAMP genes are regulated by IL-1β and 1,25D in SAECs.(A) Expression of DEFB4 and CAMP in SAECs as measured by RT/qPCR. Cells were incubated with IL-1β (10 ng/ml) or 1,25D (100 nM) for 24 h. (B) Media supernatants from cells in (A) were tested for DEFB4 and LL-37 protein secretion by ELISA. (C) and (D) Expression of DEFB4 (C) and CAMP (D) genes in SAECs incubated with conditioned media from uninfected (NI) or H37Rv-infected (I) THP-1 cells treated with vehicle or 100 nM 1,25D (+D) for 24 hours. Media was treated with neutralizing antibody against IL-1β (α-IL-1β), or normal serum IgG, as indicated, for 30 minutes prior to incubation with cells. Values are expressed as a fold of the NI control. All data are from one experiment and representative of three independent experiments using separate donors of SAECs (n = 3, mean, s.d.). *P<0.05, **P<0.01 as determined by Student's t-test relative untreated (A, B) or respective IgG (C) control.
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ppat-1003407-g004: DEFB4 and CAMP genes are regulated by IL-1β and 1,25D in SAECs.(A) Expression of DEFB4 and CAMP in SAECs as measured by RT/qPCR. Cells were incubated with IL-1β (10 ng/ml) or 1,25D (100 nM) for 24 h. (B) Media supernatants from cells in (A) were tested for DEFB4 and LL-37 protein secretion by ELISA. (C) and (D) Expression of DEFB4 (C) and CAMP (D) genes in SAECs incubated with conditioned media from uninfected (NI) or H37Rv-infected (I) THP-1 cells treated with vehicle or 100 nM 1,25D (+D) for 24 hours. Media was treated with neutralizing antibody against IL-1β (α-IL-1β), or normal serum IgG, as indicated, for 30 minutes prior to incubation with cells. Values are expressed as a fold of the NI control. All data are from one experiment and representative of three independent experiments using separate donors of SAECs (n = 3, mean, s.d.). *P<0.05, **P<0.01 as determined by Student's t-test relative untreated (A, B) or respective IgG (C) control.
Mentions: Upon infection, alveolar macrophages initially phagocytose Mtb. Importantly, alveolar macrophages are also in direct contact with the respiratory epithelial surface [4]. Although the immunological contribution of lung epithelia is well studied in other contexts [52], it is poorly characterized during the course of Mtb infection. IL-1β induces expression of genes encoding AMPs in epithelial cells through its capacity to stimulate the activity of transcription factor, NF-κB, leading to secretion of antimicrobial proteins [53], [54]. Thus, to understand the potential crosstalk between alveolar macrophages and the human respiratory epithelial surface, we conducted a series of experiments using an in vitro co-culture system between macrophages and human primary cultures of non-polarized small airway epithelial cells (SAECs) from multiple donors. In cultures of SAECs alone, recombinant IL-1β induced expression of the gene encoding DEFB4, while 1,25D had no significant effect (Figure 4A). Conversely, induction of CAMP gene expression was largely 1,25D-dependent (Figure 4A). Media supernatants from these treated SAECs were assayed for DEFB4 and CAMP secretion by ELISA. DEFB4 was detected under control conditions, and was found to be elevated when SAECs were treated with rIL-1β, but not 1,25D (Figure 4B). In contrast, the cleaved C-terminal of CAMP, LL-37, was not detected above background signal under any of the conditions (Figure 4B). In comparison, neither DEFB4 nor LL-37 was detected in media supernatants from THP-1 cells infected with Mtb using this assay (data not shown).

Bottom Line: We found that the virulent Mtb strain H37Rv elicits a broad host transcriptional response.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.

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