IFN-γ- and TNF-Independent Vitamin D-Inducible Human Suppression of Mycobacteria: The Role of Cathelicidin LL-37

Adrian R. Martineau, Katalin A. Wilkinson, Sandra M. Newton, R. Andres Floto, Anthony W. Norman, Keira Skolimowska, Robert N. Davidson, Ole E. Sørensen, Beate Kampmann, Christopher J. Griffiths and Robert J. Wilkinson
J Immunol June 1, 2007, 178 (11) 7190-7198; DOI: https://doi.org/10.4049/jimmunol.178.11.7190

Article Figures & Data

Figures

  • FIGURE 1.
    FIGURE 1.

    Ability of 1α,25(OH)2D3 to suppress the growth of BCG-lux. A, PBMC of 10 healthy donors were inoculated with a fixed RLU content of BCG-lux. The growth of BCG over the following 96 h in the presence of varying amounts of 1α,25(OH)2D3 was determined by dividing the luminescence at 96 h by that at t = 0 to give a LR for each donor. 1α,25(OH)2D3 was associated with a dose-dependent reduction in luminescence (•) that became statistically significant at 10−8 M. This effect was dependent on the presence of cells because 1α,25(OH)2D3 had no affect in their absence (○). B, PBMC of 10 donors were cultured with BCG-lux and MTB-lux in the presence or absence of 10−6 M 1α,25(OH)2D3. 1α,25(OH)2D3 induced a fall in the growth of BCG-lux from 1.74 × 106 to 9.66 × 105 RLU/ml and a fall in MTB-lux from 1.05 × 106 to 5.81 × 105 RLU/ml (p = 0.002 and <0.0001, respectively). C, PBMC of 6 donors were cultured with MTB-lux for 96 h in the presence or absence of 10−6 M 1α,25(OH)2D3. Significant suppression of the CFU of MTB was seen in all donors with the mean decreasing from 8.83 × 104 to 5.51 × 104 CFU/ml (p = 0.031). A similar decrease in luminescence in the same cultures was also observed (p = 0.063). Error bars, SE.

  • FIGURE 2.
    FIGURE 2.

    The protective effect of 1α,25(OH)2D3 against mycobacteria requires nuclear signaling. A, The selective nuclear VDR agonist V (•) was associated with dose-dependent suppression of BCG-lux in PBMC culture that was evident at a concentration as low as 10−9 M, whereas the membrane-specific agonist JN (▵) had no affect. B, In four donors, 10−8 M 1α,25(OH)2D3 significantly suppressed BCG-lux (p < 0.001). Increasing concentrations of the VDRmem antagonist HL were unable to reverse this suppression. C, In six additional donors, MK (a partial antagonist of the nuclear VDR) had moderate agonist effects at 10−7 M. The antagonist activity of MK was revealed by complete reversal of the suppressive effect of 10−8 M 1α,25(OH)2D3 (p = 0.016 by comparison with cultures that contained 1α,25(OH)2D3 alone). Error bars show SE.

  • FIGURE 3.
    FIGURE 3.

    Cytokine secretion and HLA-DR expression in cells cocultured with BCG-lux and 1α,25(OH)2D3. The BCG-lux induced secretion of IFN-γ and IL-12p40 at 96 h, and TNF at 24 h in the presence of varying concentrations of 1α,25(OH)2D3 in culture supernatants from 13 donors was assayed. A–C, There was dose-dependent 1α,25(OH)2D3 suppression of IFN-γ, IL-12p40, and TNF secretion that became significant for all three cytokines at 10−9 M (p ≤ 0.008). D, In a subset of four donors, the effect of 1α,25(OH)2D3 on HLA-DR expression was determined. There was a trend toward decreased expression of HLA-DR (p = 0.125). Error bars, SE.

  • FIGURE 4.
    FIGURE 4.

    The PBMC and MN of 10 donors were stimulated with MTB in the presence or absence of 1α,25(OH)2D3 (D) for 6 or 24 h, followed by RNA extraction and quantitative RT-PCR. A, 1α,25(OH)2D3 decreased constitutive IL-12p40 in MN over the initial 72 h (p = 0.03). MTB strongly up-regulated (21- to 117-fold) IL-12p40 in MN (p ≤ 0.011). This was reversed (a 17,660-fold decrease at t = 6 h) by the continued presence of 1α,25(OH)2D3 (p < 0.0001). B, 1α,25(OH)2D3 decreased constitutive IFN-γ expression in both MN and PBMC during the 72-h preinfection culture (p ≤ 0.03). MTB strongly up-regulated (55- to 724-fold) IFN-γ in both cell types at both time points (p ≤ 0.0004). This up-regulation was reversed (130- to 1,778-fold decrease) by the continued presence of 1α,25(OH)2D3 (p ≤ 0.0001). C, MTB also up-regulated TNF (9- to 112-fold, p ≤ 0.01) in both cell types at both time points. This up-regulation was reversed (22- to 369-fold decrease) by the presence of 1α,25(OH)2D3 (p < 0.0001). D, 1α,25(OH)2D3 moderately increased the constitutive expression of IRGC in MN (2.1-fold, p = 0.017) and conversely decreased by 2.9-fold IRGC expression in MTB-stimulated PBMC at 6 h (p = 0.04). No other effect attained statistical significance. E, 1α,25(OH)2D3 increased the constitutive expression of NOS2A 3.9-fold in PBMC (p = 0.0008). In MTB-stimulated PBMC at 24 h, the mean expression of NOS2A was 5.25-fold increased (p = 0.005). Error bars, SE. The y-axis is the mean log10 fold induction.

  • FIGURE 5.
    FIGURE 5.

    1α,25(OH)2D3-mediated suppression of BCG-lux is only slightly impaired by inhibition of NO. PBMC from four donors were set up with BCG-lux in the presence of l-NMMA (5 mM) with or without 10−8 M 1α,25(OH)2D3. l-NMMA increased (by an average of 58%) the 96-h LR. 1α,25(OH)2D3 decreased the luminescence by 41%. However, the addition of l-NMMA to the 1α,25(OH)2D3-stimulated cultures resulted only in a modest (25%) increase in RLU per milliliter. Error bars, SE.

  • FIGURE 6.
    FIGURE 6.

    Regulation of the cathelicidin hCAP18 by 1α,25(OH)2D3 and its effect on protein production. A, PBMC and MN of 10 donors were set up and RNA extracted as described previously. 1α,25(OH)2D3 increased constitutive cathelicidin gene expression 50- to 206-fold in both MN and PBMC over the initial 72 h (p ≤ 0.002). In MN, MTB down-regulated (4.59- to 13.7-fold) cathelicidin at 6 and 24 h (p ≤ 0.015). 1α,25(OH)2D3 (10−6 M) completely reversed (527- to 774-fold increase) this MTB-mediated suppression of cathelicidin (p ≤ 0.0001). B, Confocal microscopy of PBMC cultured in the presence or absence of 10−6 M 1α,25(OH)2D3 for 72 h. Immunostaining for hCAP-18 is shown in red and CD14 in green. Some hCAP-18 colocalized with CD14 in 1α,25(OH)2D3-stimulated cells. No staining for hCAP-18 was observed when preimmune rabbit IgG was substituted for anti-hCAP-18 antiserum, indicating that binding of primary Ab was specific. Optical sectioning at 1 μM intervals revealed positive staining for hCAP-18 in a granular distribution, with more diffuse staining for CD14. These granular areas of positive hCAP-18 staining were not continuous between sections, indicating that some hCAP-18 is located in the intracytoplasmic compartment.

  • FIGURE 7.
    FIGURE 7.

    Effect of synthetic cathelicidin LL-37 on the growth of MTB. A, LL-37 led to a dose-dependent reduction in the growth of MTB in broth culture that became significant at 2 μg/ml (p = 0.039) and was maximal (75.7% reduction) at 200 μg/ml. The mean of six replicates is shown with or without SE. B, The effect of low-dose (5 μg/ml) LL-37 on MTB under iron-limiting conditions. At 192 h, the CFU recovered from cultures containing LL-37 and 10−8 M free iron were ∼2-fold reduced (2.42 × 106 vs 4.96 × 106, p < 0.0001) when compared with cultures that contained LL-37 in the presence of 2 μM iron. Mean of 12 replicates ± SE.

Tables

  • Table I.

    Vitamin D analogs used in the study

    AnalogNameActionReference
    JN1α,25(OH)2lumisterol36-Cis-locked agonist with poor transcriptional activity but rapid membrane(49 )
    acting action
    V1,25-(OH)2-16-ene-23-yne-D3Specific nuclear agonist, ∼200- to 500-fold more active than 1α,25(OH)2D3(50 )
    MK23S-25-dehydro-1 α,25(OH)D3-26,23-lactoneNuclear antagonist(51 )
    HL1β,25(OH)2D3Membrane antagonist(52 )
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