Vitamin D is required for IFN-gamma-mediated antimicrobial activity of human macrophages

Abstract

Control of tuberculosis worldwide depends on our understanding of human immune mechanisms, which combat the infection. Acquired T cell responses are critical for host defense against microbial pathogens, yet the mechanisms by which they act in humans remain unclear. We report that T cells, by the release of interferon-γ (IFN-γ), induce autophagy, phagosomal maturation, the production of antimicrobial peptides such as cathelicidin, and antimicrobial activity against Mycobacterium tuberculosis in human macrophages via a vitamin D-dependent pathway. IFN-γ induced the antimicrobial pathway in human macrophages cultured in vitamin D-sufficient sera, but not in sera from African-Americans that have lower amounts of vitamin D and who are more susceptible to tuberculosis. In vitro supplementation of vitamin D-deficient serum with 25-hydroxyvitamin D3 restored IFN-γ-induced antimicrobial peptide expression, autophagy, phagosome-lysosome fusion, and antimicrobial activity. These results suggest a mechanism in which vitamin D is required for acquired immunity to overcome the ability of intracellular pathogens to evade macrophage-mediated antimicrobial responses. The present findings underscore the importance of adequate amounts of vitamin D in all human populations for sustaining both innate and acquired immunity against infection.

Fig. 1

T cell–secreted IFN-γ induces the vitamin D antimicrobial pathway. (A) T_H1 and T_H2 T cell clones were stimulated with plate-bound mAb to CD3 antibody, and supernatants were collected after 18 hours. T cell supernatants were added to human monocytes for 24 hours in 10% vitamin D–sufficient human serum, and amounts of mRNA encoding the antimicrobial peptides cathelicidin (Cath.) and DEFB4 were measured by qPCR (mean fold change ± SEM; shown is one representative donor done in triplicate). In parallel, T cell supernatants were characterized according to their secreted concentrations of IFN-γ and IL-4 (protein concentrations in ng/ml ± SD). (B) The correlation between T cell supernatant cytokine concentrations (ng/ml) and monocyte antimicrobial gene expression (mean fold change) was determined and represented as the correlation coefficient r. (C) Monocytes were pretreated with mAb to IFN-γ or isotype control antibody for 15 min and then stimulated with the T_H1 T cell clone supernatant for 20 or 24 hours in 10% vitamin D–sufficient human serum. mRNA quantities were measured by qPCR (mean fold change ± SEM, n = 7 to 9). *P < 0.05.

Fig. 2

IFN-γ and TLR2/1L induce a common vitamin D–dependent antimicrobial pathway. Primary human monocytes were stimulated with rIFN-γ or TLR2/1L for 24 hours in 10% vitamin D–sufficient human serum. (A and B) Cathelicidin and DEFB4 (A) and CYP27B1 and VDR (B) gene expression was assessed by qPCR (mean fold change ± SEM, n = 3 to 7). (C) To assess CYP27b1 activity, we cultured human monocytes treated with rIFN-γ in 10% FCS overnight for an additional 5 hours with [³H]25D3. The amount of conversion to [³H]1,25D3 and [³H]24,25D3 was measured by high-performance liquid chromatography, and CYP27b1 activity was calculated as the ratio of 1,25D3/24,25D3 (relative change compared to media ± SEM, n = 4). (D) Human monocytes were transfected with siRNA oligos specific for CYP27B1 (siCYP27B1), VDR (siVDR), or nonspecific (siCTRL) and then treated with IFN-γ in 10% vitamin D–sufficient serum for 20 or 24 hours. Cathelicidin, DEFB4, and CD64 gene expression was determined by qPCR (mean fold change ± SEM, n = 3 to 5). (E) Human monocytes were cultured in 10% vitamin D–sufficient human serum, pretreated with the VDR antagonist VAZ (ZK159222) for 15 min, and then exposed to rIFN-γ. Cathelicidin, DEFB4, and CD64 gene expression was determined by qPCR (mean fold change ± SEM, n = 4 to 7). *P ≤ 0.05.

Fig. 3

IFN-γ induction of the vitamin D antimicrobial pathway is IL-15–dependent. (A) IL-15 cell surface expression on monocytes stimulated with rIFN-γ or TLR2/1L as measured by flow cytometry at 24 hours [Δ mean fluorescence intensity (MFI) ± SEM, n = 3]. (B) Wild-type (WT), MyD88^−/−, and STAT1^−/− bone marrow–derived macrophages (BMDMs) were stimulated with murine rIFN-γ or TLR2/1L for 4 hours. IL-15 mRNA was quantified by qPCR (mean fold change ± SEM, n = 4). (C) Primary human monocytes were incubated with anti–IL-15 mAb, isotype, or media control for 15 min and stimulated with human rIFN-γ in 10% FCS for 24 hours. CYP27B1 gene expression was assessed by qPCR (mean fold change ± SEM, n = 5). (D) Monocytes were cultured as described in (C) in 10% vitamin D–sufficient human serum for 20 or 24 hours. Cathelicidin and DEFB4 gene expression was assessed by qPCR (mean fold change ± SEM, n = 6 to 8). *P ≤ 0.05.

Fig. 4

IFN-γ–induced autophagy and phagosome maturation are VDR-dependent. (A) Human primary monocytes were incubated with rIFN-γ, rapamycin (Rapa), or medium for 24 hours in 10% vitamin D–sufficient human serum, fixed, and immunolabeled with anti–LC3-FITC (fluorescein isothiocyanate) antibody (green). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Representative immunofluorescence images are shown. (B) LC3 punctate cells were quantified (mean of percent positive cells ± SEM, n = 5). (C) Monocytes were incubated with rIFN-γ in the presence or absence of 3-MA or WM for 24 hours, and the percentage of LC3 punctate cells was determined (mean ± SEM, n = 4). (D) Gene expression was knocked down inmonocytes with lentiviral shRNA specific for Beclin-1, Atg5, or control shRNA followed by stimulation with rIFN-γ for 24 hours. Percent LC3-positive cells was determined (mean ± SEM, n = 4). (E) Monocytes were incubated with rIFN-γ in the presence or absence of VAZ (1, 10, or 100 nM) for 24 hours and labeled as in (A). Representative immunofluorescence pictures of LC3 punctate cells are shown. IPA, isopropyl alcohol. (F) LC3 punctate cells were quantified (mean of percent positive cells ± SEM, n = 5). (G) Monocytes were stimulated with IFN-γ for 24 hours in 10% vitamin D–sufficient human serum, loaded with LysoTracker (green), and stained for LC3 (red). Representative immunofluorescence images of three independent replicates are shown. TRITC, tetramethylrhodamine isothiocyanate. (H) Human MDMs were infected with FITC–M. tuberculosis (green) for 4 hours, washed, and stimulated with rIFN-γ in the presence or absence of VAZ (1, 10, or 100 nM) for 30 hours. Lysosomes were stained with LysoTracker (red), and cells were fixed. Representative fluorescence-merged images are shown. (I) Quantitative analysis for (H) (mean ± SEM, n = 6). *P < 0.05; **P < 0.01.

Fig. 5

IFN-γ induction of antimicrobial response is dependent on the concentration of serum vitamin D. (A) Concentrations of 25D of individual sera obtained from white (n = 4) and African-American (n = 4) donors were measured (mean 25D serum amounts ± SEM). Monocytes were cultured with 10% serum from either white or African-American individuals and stimulated with IFN-γ for 20 hours. Cathelicidin and DEFB4 gene expression was determined by qPCR (mean fold change ± SEM). (B) Monocytes were cultured in vitamin D–sufficient (25D = 98 nM) or vitamin D–deficient (25D = 45 nM) pooled human serum (HuS) and stimulated with rIFN-γ for 20 or 24 hours. Cathelicidin and DEFB4 gene expression was determined by qPCR (mean fold change ± SEM, n = 3 to 4). (C) Monocytes were cultured in 10% vitamin D–deficient serum (25D = 45 nM), with or without the addition of 25D3 to reach sufficient concentrations, and stimulated with rIFN-γ.Concentrations of cathelicidin and DEFB4 gene expression were measured at 20 or 24 hours (mean fold change ± SEM, n = 3 to 4). (D) MDMs were cultured in 10% vitamin D–sufficient serum (25D = 98 nM) or vitamin D–deficient serum (25D = 45 nM), with or without the addition of 25D3 to reach sufficient concentrations, and stimulated with rIFN-γ. Cathelicidin and DEFB4 gene expression was measured at 20 to 24 hours (mean fold change ± SEM, n = 5 to 6). (E and F) Primary human monocytes (E) and MDMs (F) were infected with M. tuberculosis H37Rv and cultured with medium or rIFN-γ in 10% vitamin D–sufficient (25D = 98 nM) or vitamin D–deficient (25D = 45nM) human serum. Viable bacteria were quantified by CFU assay after days 0 and 3 (E) or days 0 and 5 (F) (mean ± SEM, n = 9). *P < 0.05; **P < 0.01; ***P < 0.001. ns, not significant.

Fig. 6

IFN-γ induces an antimicrobial pathway in human monocytes/macrophages. This model shows that STAT1-dependent induction of IL-15 by IFN-γ leads to the up-regulation of VDR and CYP27b1. CYP27b1 hydroxylates the inactive form of vitamin D (25D) into the active form (1,25D), which mediates the up-regulation of antimicrobial peptides cathelicidin and DEFB4. The intracrine-produced 1,25D also triggers autophagy, which overcomes the M. tuberculosis–induced phagosome maturation block, leading to autophagolysosomal fusion and antimicrobial activity against M. tuberculosis.