Vitamin D rescues impaired Mycobacterium tuberculosis-mediated tumor necrosis factor release in macrophages of HIV-seropositive individuals through an enhanced Toll-like receptor signaling pathway in vitro

Abstract

Mycobacterium tuberculosis disease represents an enormous global health problem, with exceptionally high morbidity and mortality in HIV-seropositive (HIV(+)) persons. Alveolar macrophages from HIV(+) persons demonstrate specific and targeted impairment of critical host cell responses, including impaired M. tuberculosis-mediated tumor necrosis factor (TNF) release and macrophage apoptosis. Vitamin D may promote anti-M. tuberculosis responses through upregulation of macrophage NO, NADPH oxidase, cathelicidin, and autophagy mechanisms, but whether vitamin D promotes anti-M. tuberculosis mechanisms in HIV(+) macrophages is not known. In the current study, human macrophages exposed to M. tuberculosis demonstrated robust release of TNF, IκB degradation, and NF-κB nuclear translocation, and these responses were independent of vitamin D pretreatment. In marked contrast, HIV(+) U1 human macrophages exposed to M. tuberculosis demonstrated very low TNF release and no significant IκB degradation or NF-κB nuclear translocation, whereas vitamin D pretreatment restored these critical responses. The vitamin D-mediated restored responses were dependent in part on macrophage CD14 expression. Importantly, similar response patterns were observed with clinically relevant human alveolar macrophages from healthy individuals and asymptomatic HIV(+) persons at high clinical risk of M. tuberculosis infection. Taken together with the observation that local bronchoalveolar lavage fluid (BALF) levels of vitamin D are severely deficient in HIV(+) persons, the data from this study demonstrate that exogenous vitamin D can selectively rescue impaired critical innate immune responses in vitro in alveolar macrophages from HIV(+) persons at risk for M. tuberculosis disease, supporting a potential role for exogenous vitamin D as a therapeutic adjuvant in M. tuberculosis infection in HIV(+) persons.

Fig 1

1,25D₃ rescues M. tuberculosis-mediated TNF release in HIV⁺ human macrophages. Differentiated U937 (A) and HIV⁺ U1 (B and C) macrophages were incubated with irradiated virulent M. tuberculosis (MTb) (MOI of 10:1 for 24 h) in the presence or absence of 1,25D₃ (VD) pretreatment (24 h). TNF in cell culture supernatants was measured by ELISA (R&D). Figures are representative of individual experiments with similar results (n = 6). Quantitative data represent means ± standard errors of the means (SEM). US, unstimulated. *, P < 0.05.

Fig 2

1,25D₃ enhances TNF transcription in HIV⁺ human macrophages. (A) RT-PCR and real-time PCR for VDR were performed on total RNAs from differentiated U937 and HIV⁺ U1 macrophages (n = 4). (B) Differentiated U937 and HIV⁺ U1 cells were incubated with M. tuberculosis or BCG (M. bovis) (MOI, 10:1) for 3 h in the presence or absence of 1,25D₃ pretreatment (24 h). TNF mRNA was measured by real-time PCR (n = 4). Quantitative data represent means ± SEM. *, P < 0.05; **, P < 0.01.

Fig 3

1,25D₃ increases TLR signaling but not TLR expression in HIV⁺ U1 macrophages. (A and B) Differentiated human U937 and HIV⁺ U1 macrophages were incubated with the TLR ligands lipid A (LA) (for TLR4), PamCys (for TLR2/1), and 19-kDa lipoprotein from M. tuberculosis (19 kDa MTb; 1 μg/ml) (for TLR2/1) for 24 h in the presence or absence of 1,25D₃ pretreatment. Cell-free culture supernatants were assayed for TNF by ELISA (n = 3). (C) Differentiated U937 and HIV⁺ U1 macrophages were incubated for 24 h in the presence or absence of 1,25D₃ pretreatment and then stained with PE-labeled anti-TLR antibodies or isotype control antibody. Surface expression was measured by flow cytometry. Left panels show isotype control (gray lines)- and TLR (black lines)-labeled cells; right panels show TLR-labeled cells (gray lines) and 1,25D₃-treated TLR-labeled cells (black lines). Representative histograms for independent experiments with similar results (n = 3) are shown. (D) Specific TLR2 and TLR4 mRNAs were detected by real-time PCR (n = 3). Quantitative data represent means ± SEM. *, P < 0.05.

Fig 4

1,25D₃ upregulates NF-κB signaling in HIV⁺ human macrophages. (A) Differentiated U937 and HIV⁺ U1 macrophages were incubated with M. tuberculosis (MOI, 10:1) for 0 to 120 min in the presence or absence of 1,25D₃ and PDTC. Cell lysates were resolved by Western blotting using a specific antibody to IκBα. Representative blots for three independent experiments with similar results are shown. Quantitative densitometric analyses of IκBα bands are displayed directly beneath the blots. (B) NF-κB nuclear translocation in nuclear extracts was measured by ELISA (n = 3). Quantitative data represent means ± SEM. *, P < 0.05.

Fig 5

1,25D₃ induces CD14 expression in human macrophages. (A) Differentiated U937 and HIV⁺ U1 macrophages were incubated for 24 h in the presence or absence of 1,25D₃ pretreatment and then stained with PE-labeled anti-CD14 antibody or isotype control antibody. Surface expression was measured by flow cytometry. Left panels show isotype control (gray lines)- and CD14 (black lines)-labeled cells; right panels show CD14-labeled cells (gray lines) and 1,25D₃-treated CD14-labeled cells (black lines). Representative histograms from individual experiments with similar results (n = 3) are shown. (B and C) Differentiated U937 (B) and HIV⁺ U1 (C) macrophages were treated with M. tuberculosis (MOI, 10:1) or LPS (1 μg/ml) in the presence or absence of vitamin D pretreatment (24 h) and the indicated antibodies. TNF in cell culture supernatants was measured by ELISA (R&D). The data are representative of three individual experiments with similar results. Quantitative data represent means ± SEM. *, P < 0.05.

Fig 6

1,25D₃ rescues M. tuberculosis-mediated TNF release in human alveolar macrophages from HIV⁺ persons. (A) AMs from healthy (n = 6) and HIV⁺ (n = 2) persons were incubated with M. tuberculosis or BCG (MOI of 10:1 for 24 h) in the presence or absence of 1,25D₃ pretreatment (24 h). TNF in cell culture supernatants was measured by ELISA (R&D). (B) Specific TLR2 and TLR4 mRNAs were detected by real-time PCR (n = 3). (C) AMs were incubated for 24 h in the presence or absence of 1,25D₃ pretreatment and then stained with PE-labeled anti-TLR or isotype control antibody. Surface expression was measured by flow cytometry. Left panels show isotype control (gray lines)- and receptor (black lines)-labeled cells; right panels show receptor-labeled cells (gray lines) and 1,25D₃-treated receptor-labeled cells (black lines). Representative histograms for individual experiments with similar results (n = 3 for healthy individuals and 2 for HIV⁺ individuals) are shown. Quantitative data represent means ± SEM. *, P < 0.05; **, P < 0.01.

Fig 7

1,25D₃ rescue of M. tuberculosis-mediated TNF release in human alveolar macrophages from HIV⁺ persons is dependent on CD14. (A) AMs were incubated for 24 h in the presence or absence of 1,25D₃ pretreatment and then stained with PE-labeled anti-CD14 or isotype control antibody. Surface expression was measured by flow cytometry. Left panels show isotype control (gray lines)- and receptor (black lines)-labeled cells; right panels show receptor-labeled cells (gray lines) and 1,25D₃-treated receptor-labeled cells (black lines) (n = 3). (B) HIV⁺ AMs were treated with M. tuberculosis (MOI of 0.25:1), BCG (MOI of 10:1), or LPS (1 μg/ml) in the presence or absence of 1,25D₃ pretreatment (24 h) and the indicated antibodies. TNF in cell culture supernatants was measured by ELISA (n = 2). (C) 25D₃ levels were measured in the cell-free BALF of healthy and HIV-infected Indian patients with and without active M. tuberculosis infection (for HIV⁻ M. tuberculosis⁻ individuals, n = 38; for HIV⁻ M. tuberculosis⁺ individuals, n = 35; for HIV⁺ M. tuberculosis⁻ individuals, n = 12; and for HIV⁺ M. tuberculosis⁺ individuals, n = 17) by ELISA. Quantitative data represent means ± SEM. *, P < 0.05.