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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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Co-culture with SAECs enhances survival of Mtb-infected macrophages.(A) Schematic representation in profile of co-culture system. THP-1 cells were cultured and infected in the lower well and SAECs were seeded in the upper transwell bucket. (B) LDH activity measured in the media supernatant from macrophages (Mφ) infected with H37Rv at an MOI of 5 or 10, as indicated, and treated with vehicle control or 100 nM 1,25D (+D), with and without the presence of SAECs in transwell co-culture (CC). Signal was normalized to spontaneous LDH release levels from uninfected macrophages, as measured from media supernatant collected from cells at each day. All data is expressed relative to LDH activity of the Mφ condition infected at an MOI of 5 at 24 hours post-infection. Data are from one experiment and representative of two independent experiments using separate donors of SAECs (n = 3, mean, s.d.). **P<0.01 as determined by two-way ANOVA comparing Mφ to CC conditions. (C) Representative phase-contrast microscopy of macrophages 3 days after infection with and without 1,25D treatment in the presence or absence of SAECs in co-culture. In the macrophage only condition, most of the cells visible appear round and out of focus as they are floating freely in the media, while cells co-cultured with SAECs are almost all adherent and exhibit normal macrophage morphology.
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ppat-1003407-g005: Co-culture with SAECs enhances survival of Mtb-infected macrophages.(A) Schematic representation in profile of co-culture system. THP-1 cells were cultured and infected in the lower well and SAECs were seeded in the upper transwell bucket. (B) LDH activity measured in the media supernatant from macrophages (Mφ) infected with H37Rv at an MOI of 5 or 10, as indicated, and treated with vehicle control or 100 nM 1,25D (+D), with and without the presence of SAECs in transwell co-culture (CC). Signal was normalized to spontaneous LDH release levels from uninfected macrophages, as measured from media supernatant collected from cells at each day. All data is expressed relative to LDH activity of the Mφ condition infected at an MOI of 5 at 24 hours post-infection. Data are from one experiment and representative of two independent experiments using separate donors of SAECs (n = 3, mean, s.d.). **P<0.01 as determined by two-way ANOVA comparing Mφ to CC conditions. (C) Representative phase-contrast microscopy of macrophages 3 days after infection with and without 1,25D treatment in the presence or absence of SAECs in co-culture. In the macrophage only condition, most of the cells visible appear round and out of focus as they are floating freely in the media, while cells co-cultured with SAECs are almost all adherent and exhibit normal macrophage morphology.

Mentions: Next, to model the consequences of the real-time interaction between infected macrophages and the alveolar epithelia during the initial round of infection, we established a transwell co-culture system between Mtb-infected macrophages and SAECs (Figure 5A). Macrophages were infected with Mtb for 4 hours and washed extensively to eliminate any remaining extracellular mycobacteria. A transwell bucket containing co-cultured lung epithelial cells was then added to the tissue culture plate containing the infected macrophages. The two cell populations were separated by a 0.4 µm filter to allow for the exchange of secreted proteins but prevent migration of mycobacteria. Note that in control experiments no mycobacteria were detected in the transwell bucket 4 days after infection (data not shown). Co-culture of macrophages infected at an multiplicity of infection (MOI) of 5 with SAECs dramatically extended macrophage cell survival at three days post-infection, as measured by cytoplasmic lactate dehydrogenase (LDH) release (Figure 5B), a marker of plasma-membrane compromise and necrosis. After 3 days of infection, the relative amount of LDH release was comparable to that seen 24 hours after infection under macrophage-only conditions. 1,25D treatment had no effect on the survival of infected cells cultured in the absence or presence of SAECs. When macrophages infected at an MOI of 10 were co-cultured with SAECs, LDH release was slightly higher, but a similar protective effect of SAECs was observed (Figure 5B). The protective benefit of the SAECs in co-culture was also clear by the relative amount of adherent cells remaining as visualized by phase-contrast microscopy (Figure 5C); most of the macrophages co-cultured with SAECs were still adherent, whereas infected macrophages cultured in the absence of SAECs had detached from the plate.


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)

Co-culture with SAECs enhances survival of Mtb-infected macrophages.(A) Schematic representation in profile of co-culture system. THP-1 cells were cultured and infected in the lower well and SAECs were seeded in the upper transwell bucket. (B) LDH activity measured in the media supernatant from macrophages (Mφ) infected with H37Rv at an MOI of 5 or 10, as indicated, and treated with vehicle control or 100 nM 1,25D (+D), with and without the presence of SAECs in transwell co-culture (CC). Signal was normalized to spontaneous LDH release levels from uninfected macrophages, as measured from media supernatant collected from cells at each day. All data is expressed relative to LDH activity of the Mφ condition infected at an MOI of 5 at 24 hours post-infection. Data are from one experiment and representative of two independent experiments using separate donors of SAECs (n = 3, mean, s.d.). **P<0.01 as determined by two-way ANOVA comparing Mφ to CC conditions. (C) Representative phase-contrast microscopy of macrophages 3 days after infection with and without 1,25D treatment in the presence or absence of SAECs in co-culture. In the macrophage only condition, most of the cells visible appear round and out of focus as they are floating freely in the media, while cells co-cultured with SAECs are almost all adherent and exhibit normal macrophage morphology.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675149&req=5

ppat-1003407-g005: Co-culture with SAECs enhances survival of Mtb-infected macrophages.(A) Schematic representation in profile of co-culture system. THP-1 cells were cultured and infected in the lower well and SAECs were seeded in the upper transwell bucket. (B) LDH activity measured in the media supernatant from macrophages (Mφ) infected with H37Rv at an MOI of 5 or 10, as indicated, and treated with vehicle control or 100 nM 1,25D (+D), with and without the presence of SAECs in transwell co-culture (CC). Signal was normalized to spontaneous LDH release levels from uninfected macrophages, as measured from media supernatant collected from cells at each day. All data is expressed relative to LDH activity of the Mφ condition infected at an MOI of 5 at 24 hours post-infection. Data are from one experiment and representative of two independent experiments using separate donors of SAECs (n = 3, mean, s.d.). **P<0.01 as determined by two-way ANOVA comparing Mφ to CC conditions. (C) Representative phase-contrast microscopy of macrophages 3 days after infection with and without 1,25D treatment in the presence or absence of SAECs in co-culture. In the macrophage only condition, most of the cells visible appear round and out of focus as they are floating freely in the media, while cells co-cultured with SAECs are almost all adherent and exhibit normal macrophage morphology.
Mentions: Next, to model the consequences of the real-time interaction between infected macrophages and the alveolar epithelia during the initial round of infection, we established a transwell co-culture system between Mtb-infected macrophages and SAECs (Figure 5A). Macrophages were infected with Mtb for 4 hours and washed extensively to eliminate any remaining extracellular mycobacteria. A transwell bucket containing co-cultured lung epithelial cells was then added to the tissue culture plate containing the infected macrophages. The two cell populations were separated by a 0.4 µm filter to allow for the exchange of secreted proteins but prevent migration of mycobacteria. Note that in control experiments no mycobacteria were detected in the transwell bucket 4 days after infection (data not shown). Co-culture of macrophages infected at an multiplicity of infection (MOI) of 5 with SAECs dramatically extended macrophage cell survival at three days post-infection, as measured by cytoplasmic lactate dehydrogenase (LDH) release (Figure 5B), a marker of plasma-membrane compromise and necrosis. After 3 days of infection, the relative amount of LDH release was comparable to that seen 24 hours after infection under macrophage-only conditions. 1,25D treatment had no effect on the survival of infected cells cultured in the absence or presence of SAECs. When macrophages infected at an MOI of 10 were co-cultured with SAECs, LDH release was slightly higher, but a similar protective effect of SAECs was observed (Figure 5B). The protective benefit of the SAECs in co-culture was also clear by the relative amount of adherent cells remaining as visualized by phase-contrast microscopy (Figure 5C); most of the macrophages co-cultured with SAECs were still adherent, whereas infected macrophages cultured in the absence of SAECs had detached from the plate.

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