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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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Control of Mtb infection by co-cultured epithelial cells is dependent on epithelial IL1R1 and DEFB4 expression.(A) CFU of macrophages (Mφ) infected with H37Rv at an MOI of 5 for 4 hours and treated with vehicle control or 100 nM 1,25D (+D), with and without the additional presence of SAECs in transwell co-culture (CC). Data are from three experimental replicates (mean and SD) and representative of three independent experiments using different donors of primary cells. Statistical significance was determined by one-way ANOVA. (*P<0.05). (B) Validation of siRNA-mediated knockdown of IL1R1 expression in SAECs. RNA was extracted from SAECs cells 3 days after the initiation of co-culture and 4.5 days after transfection of control siRNA (siCTL) or siIL1R1. Data are from three experimental replicates (mean and SD), **P<0.01 as determined by student's T-test relative to respective siCTL controls. (C) CFU quantification of Mtb in THP-1 cells infected at an MOI of 5 for 4 hours after 72 hours of co-culture with SAEC cells transfected with control siRNA (siCTL) or siRNA specific to IL1R1. 1,25D (D) was added as indicated. Data are from three experimental replicates (mean and SD) and representative of two independent experiments. Statistical significance was determined by one-way ANOVA. (**P<0.01). (D) Validation of siRNA-mediated knockdown of DEFB4 expression in SAECs. RNA was extracted from SAECs cells 3 days after the initiation of co-culture and 4.5 days after transfection of control siRNA (siCTL) or siDEFB4. Data are from three experimental replicates (mean and SD) and representative to two independent replicates. **P<0.01 as determined by student's T-test relative to siCTL control. (E) CFU quantification of Mtb in THP-1 cells infected at an MOI of 5 for 4 hours after 72 hours of co-culture with SAEC cells transfected with control siRNA (siCTL) or siRNA specific to IL1R1 or DEFB4. Data are from three experimental replicates (mean and SD) and representative of two independent experiments using separate donors of SAECs. Statistical significance was determined by one-way ANOVA. (*P<0.05).
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ppat-1003407-g006: Control of Mtb infection by co-cultured epithelial cells is dependent on epithelial IL1R1 and DEFB4 expression.(A) CFU of macrophages (Mφ) infected with H37Rv at an MOI of 5 for 4 hours and treated with vehicle control or 100 nM 1,25D (+D), with and without the additional presence of SAECs in transwell co-culture (CC). Data are from three experimental replicates (mean and SD) and representative of three independent experiments using different donors of primary cells. Statistical significance was determined by one-way ANOVA. (*P<0.05). (B) Validation of siRNA-mediated knockdown of IL1R1 expression in SAECs. RNA was extracted from SAECs cells 3 days after the initiation of co-culture and 4.5 days after transfection of control siRNA (siCTL) or siIL1R1. Data are from three experimental replicates (mean and SD), **P<0.01 as determined by student's T-test relative to respective siCTL controls. (C) CFU quantification of Mtb in THP-1 cells infected at an MOI of 5 for 4 hours after 72 hours of co-culture with SAEC cells transfected with control siRNA (siCTL) or siRNA specific to IL1R1. 1,25D (D) was added as indicated. Data are from three experimental replicates (mean and SD) and representative of two independent experiments. Statistical significance was determined by one-way ANOVA. (**P<0.01). (D) Validation of siRNA-mediated knockdown of DEFB4 expression in SAECs. RNA was extracted from SAECs cells 3 days after the initiation of co-culture and 4.5 days after transfection of control siRNA (siCTL) or siDEFB4. Data are from three experimental replicates (mean and SD) and representative to two independent replicates. **P<0.01 as determined by student's T-test relative to siCTL control. (E) CFU quantification of Mtb in THP-1 cells infected at an MOI of 5 for 4 hours after 72 hours of co-culture with SAEC cells transfected with control siRNA (siCTL) or siRNA specific to IL1R1 or DEFB4. Data are from three experimental replicates (mean and SD) and representative of two independent experiments using separate donors of SAECs. Statistical significance was determined by one-way ANOVA. (*P<0.05).

Mentions: To determine any effects of co-culturing with and without 1,25D on mycobacterial burden, colony forming unit (CFU) assays were performed with cells co-cultured as above. We determined changes in total Mtb burden after 72 hours of infection. The addition of SAECs resulted in a halving of mycobacterial burden at this time point, and addition of 1,25D to the co-culture system produced a further significant reduction in mycobacteria (Figure 6A). To confirm the contribution of epithelial signaling by infection- and 1,25D-induced IL-1β towards the reduction in mycobacterial burden, we knocked down IL1R1 receptor expression in SAECs 36 hours prior to their co-culture. Control experiments showed that expression of epithelial IL1R1 was reduced for at least 72 hours after siRNA-mediated knockdown (Figure 6B). Mycobacterial burden was sharply elevated 72 hours after infection in the absence or presence of 1,25D when macrophages were co-cultured with IL1R1-depleted SAECs. In contrast, co-culture of SAECs transfected with control siRNAs eliminated net mycobacterial growth in the presence of 1,25D (Figure 6C), consistent with experiments described above. To determine if epithelial secretion of DEFB4 was responsible for the increased control of mycobacterial proliferation, we transfected SAECs with either siRNA against IL1R1 or DEFB4 transcripts 36 hours prior to their co-culture with infected macrophages. Reduced expression of DEFB4 was verified by qPCR in samples collected 72 hours after the initiation of their co-culture (Figure 6D). CFU assays were performed at 72 hours after infection and demonstrated that siRNA-mediated knockdown of DEFB4 expression permitted levels of bacterial proliferation similar to what was observed in knockdown of IL1R1 (Figure 6E). Taken together, these data reveal that IL-1β secreted from infected macrophages drives a paracrine signaling cascade which contributes to control of mycobacterial burden in our culture system.


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)

Control of Mtb infection by co-cultured epithelial cells is dependent on epithelial IL1R1 and DEFB4 expression.(A) CFU of macrophages (Mφ) infected with H37Rv at an MOI of 5 for 4 hours and treated with vehicle control or 100 nM 1,25D (+D), with and without the additional presence of SAECs in transwell co-culture (CC). Data are from three experimental replicates (mean and SD) and representative of three independent experiments using different donors of primary cells. Statistical significance was determined by one-way ANOVA. (*P<0.05). (B) Validation of siRNA-mediated knockdown of IL1R1 expression in SAECs. RNA was extracted from SAECs cells 3 days after the initiation of co-culture and 4.5 days after transfection of control siRNA (siCTL) or siIL1R1. Data are from three experimental replicates (mean and SD), **P<0.01 as determined by student's T-test relative to respective siCTL controls. (C) CFU quantification of Mtb in THP-1 cells infected at an MOI of 5 for 4 hours after 72 hours of co-culture with SAEC cells transfected with control siRNA (siCTL) or siRNA specific to IL1R1. 1,25D (D) was added as indicated. Data are from three experimental replicates (mean and SD) and representative of two independent experiments. Statistical significance was determined by one-way ANOVA. (**P<0.01). (D) Validation of siRNA-mediated knockdown of DEFB4 expression in SAECs. RNA was extracted from SAECs cells 3 days after the initiation of co-culture and 4.5 days after transfection of control siRNA (siCTL) or siDEFB4. Data are from three experimental replicates (mean and SD) and representative to two independent replicates. **P<0.01 as determined by student's T-test relative to siCTL control. (E) CFU quantification of Mtb in THP-1 cells infected at an MOI of 5 for 4 hours after 72 hours of co-culture with SAEC cells transfected with control siRNA (siCTL) or siRNA specific to IL1R1 or DEFB4. Data are from three experimental replicates (mean and SD) and representative of two independent experiments using separate donors of SAECs. Statistical significance was determined by one-way ANOVA. (*P<0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675149&req=5

ppat-1003407-g006: Control of Mtb infection by co-cultured epithelial cells is dependent on epithelial IL1R1 and DEFB4 expression.(A) CFU of macrophages (Mφ) infected with H37Rv at an MOI of 5 for 4 hours and treated with vehicle control or 100 nM 1,25D (+D), with and without the additional presence of SAECs in transwell co-culture (CC). Data are from three experimental replicates (mean and SD) and representative of three independent experiments using different donors of primary cells. Statistical significance was determined by one-way ANOVA. (*P<0.05). (B) Validation of siRNA-mediated knockdown of IL1R1 expression in SAECs. RNA was extracted from SAECs cells 3 days after the initiation of co-culture and 4.5 days after transfection of control siRNA (siCTL) or siIL1R1. Data are from three experimental replicates (mean and SD), **P<0.01 as determined by student's T-test relative to respective siCTL controls. (C) CFU quantification of Mtb in THP-1 cells infected at an MOI of 5 for 4 hours after 72 hours of co-culture with SAEC cells transfected with control siRNA (siCTL) or siRNA specific to IL1R1. 1,25D (D) was added as indicated. Data are from three experimental replicates (mean and SD) and representative of two independent experiments. Statistical significance was determined by one-way ANOVA. (**P<0.01). (D) Validation of siRNA-mediated knockdown of DEFB4 expression in SAECs. RNA was extracted from SAECs cells 3 days after the initiation of co-culture and 4.5 days after transfection of control siRNA (siCTL) or siDEFB4. Data are from three experimental replicates (mean and SD) and representative to two independent replicates. **P<0.01 as determined by student's T-test relative to siCTL control. (E) CFU quantification of Mtb in THP-1 cells infected at an MOI of 5 for 4 hours after 72 hours of co-culture with SAEC cells transfected with control siRNA (siCTL) or siRNA specific to IL1R1 or DEFB4. Data are from three experimental replicates (mean and SD) and representative of two independent experiments using separate donors of SAECs. Statistical significance was determined by one-way ANOVA. (*P<0.05).
Mentions: To determine any effects of co-culturing with and without 1,25D on mycobacterial burden, colony forming unit (CFU) assays were performed with cells co-cultured as above. We determined changes in total Mtb burden after 72 hours of infection. The addition of SAECs resulted in a halving of mycobacterial burden at this time point, and addition of 1,25D to the co-culture system produced a further significant reduction in mycobacteria (Figure 6A). To confirm the contribution of epithelial signaling by infection- and 1,25D-induced IL-1β towards the reduction in mycobacterial burden, we knocked down IL1R1 receptor expression in SAECs 36 hours prior to their co-culture. Control experiments showed that expression of epithelial IL1R1 was reduced for at least 72 hours after siRNA-mediated knockdown (Figure 6B). Mycobacterial burden was sharply elevated 72 hours after infection in the absence or presence of 1,25D when macrophages were co-cultured with IL1R1-depleted SAECs. In contrast, co-culture of SAECs transfected with control siRNAs eliminated net mycobacterial growth in the presence of 1,25D (Figure 6C), consistent with experiments described above. To determine if epithelial secretion of DEFB4 was responsible for the increased control of mycobacterial proliferation, we transfected SAECs with either siRNA against IL1R1 or DEFB4 transcripts 36 hours prior to their co-culture with infected macrophages. Reduced expression of DEFB4 was verified by qPCR in samples collected 72 hours after the initiation of their co-culture (Figure 6D). CFU assays were performed at 72 hours after infection and demonstrated that siRNA-mediated knockdown of DEFB4 expression permitted levels of bacterial proliferation similar to what was observed in knockdown of IL1R1 (Figure 6E). Taken together, these data reveal that IL-1β secreted from infected macrophages drives a paracrine signaling cascade which contributes to control of mycobacterial burden in our culture system.

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