A. Human primary monocytes were incubated with LpqH in the absence or presence of neutralizing Ab for hTLR1 (αT1), hTLR2 (αT2), hTLR4 (αT4), hCD14 (αCD14) or isotype control (IC; mIgG1 and mIgG2a; shown is the data for mIgG1 Ab), and subjected to confocal analysis as described in Fig. 1A. Top, a representative image with similar results is shown (three experiments); bottom, quantification of data; LC3 punctated cells were counted manually in DAPI-stained monocytes. #P < 0.02; *P < 0.001, versus control condition. Scale bars, 5 μm.
B. Human THP-1 cells transfected with non-specific siRNA (siNS) or specific siRNAs for hTLR1 (siT1), hTLR2 (siT2), hTLR4 (siT4) or hCD14 (siCD14) were incubated in the absence or presence of LpqH (100 ng ml⁻¹) for 24 h, and subjected to confocal analysis as described in Fig. 1A. Graph is shown the percentage of LC3 punctated cells; right, RT-PCR for transfection efficiency. *P < 0.008, versus control condition. Data shown (for A and B) represent the means ± SD of three independent samples, with each experiment including at least 250 cells scored in five random fields. U, untreated and incubated.

A. Semi-quantitative RT-PCR analysis for Cyp27b1 hydroxylase and LL-37. Human monocytes were incubated with LpqH for indicated times. Results (means ± SD, three independent experiments) are normalized to β-actin expression and are presented relative to expression in LpqH-stimulated cells.
B. LL-37 expression. Human monocytes were incubated in the absence or presence of Cyp27b1 antagonist itraconazole (ITRA; 50 nM, 100 nM, 200 nM), followed by LpqH treatment. After 24 h, the cells were stained with anti-LL-37-TRITC (1:400).
C–E. THP-1 cells were transfected with non-specific siRNA (siNS) or siRNAs specific for hCyp27b1 (siCyp27b1; for C), hC/EBP-β (siC/EBPβ; for D), hTLR1 (siT1), hTLR2 (siT2), hTLR4 (siT4) or hCD14 (siCD14; for E) followed by LpqH treatment for 24 h. (C) The cells were stained with anti-VDR-TRITC (1:400). Inset, RT-PCR for transfection efficiency. (D and E) Semi-quantitative RT-PCR analysis. (E, bottom) Results (means ± SD, three independent experiments) are normalized to β-actin expression and are presented relative to expression in LpqH-stimulated cells.
F. The experimental conditions were as outlined in (B). The cells were stained with anti-LC3-FITC (1:400).
Quantitative data shown represent the means ± SD of three independent samples (B, C, F), with each experiment including at least 250 cells scored in five random fields. #P ≤ 0.03; *P ≤ 0.05; ***P ≤ 0.001, versus control conditions (A, B, C, E, F). Scales bars, 10 μm (for B and C); 5 μm (for F). U, untreated and incubated; SC, treated with solvent control (0.1% DMSO, for B and F).

A–C. Human monocytes were stimulated with LpqH (100 ng ml⁻¹) in the absence or presence of various inhibitors (AMPK inhibitor compound C; Comp C, 5, 10, 25 μM; for A) or MAPK inhibitors [p38, SB203580 (SB), 1, 5, 10 μM; ERK, U0126, 5, 10, 20 μM; JNK, SP600125 (SP), 5, 10, 20 μM; for B and C], and then subjected to Western blot (for A), confocal analysis (for B), or semi-quantitative RT-PCR analysis (for C). (B) Data shown represent the means ± SD of three independent samples, with each experiment including at least 250 cells scored in five random fields. (A and C) Representative images are shown (at least three independent experiments).
D. THP-1 cells were transfected with a C/EBP-β luciferase reporter construct. After 36 h, cells were pre-treated with MAPK inhibitors (the same conditions as shown in C), followed by LpqH stimulation for 6 h. The luciferase activity was then measured and normalized to β-galactosidase activity.
#P < 0.01; *P < 0.0004, versus control conditions. U, untreated and incubated; SC, treated with solvent control (0.1% DMSO).

A. Human primary monocytes were incubated with LpqH (50, 100, 200, 500 ng ml⁻¹; top, 100 ng ml⁻¹) in the absence or presence of human serum (HS) for 24 h, fixed, stained with DAPI to visualize the nuclei (blue), and immunolabelled with the anti-LC3 Ab, followed by the addition of FITC-conjugated goat anti-rabbit IgG (green). Above, representative immunofluorescence images (scale bars, 5 μm); below, quantification of data; LC3 punctated cells were counted manually in DAPI-stained monocytes. *P < 0.006 (Rapa, 200 ng ml⁻¹); *P < 0.002 (LpqH; 50 ng ml⁻¹); *P < 0.0001 (LpqH; 100 ng ml⁻¹); *P < 0.0004 (LpqH; 200 ng ml⁻¹), versus control conditions. Scale bar, 10 μm.
B. Quantitative analysis of the LC3 fluorescence images. Human primary monocytes transduced with lentiviruses expressing non-specific shRNA (shNS) or specific shRNAs for Beclin-1 (shBec) or Atg5 (shAtg5) were incubated in the absence or presence of LpqH (100 ng ml⁻¹) for 24 h; inset, RT-PCR for transfection efficiency. *P < 0.005, versus control conditions. Data shown (for A and B) represent the means ± SD of three independent samples, with each experiment including at least 250 cells scored in five random fields.
C. Immunoblot analyses with Abs to LC3 or β-actin for human monocytes. Representative gel images are shown (three experiments). The ratio of the intensities of the LC3-II and LC3-I bands is indicated below each lane.
D–I. Human THP-1 cells were treated with (for F–I) or without (for D, E) LpqH for 24 h and subjected to TEM analysis (representative image of two experiments). (E, G) Enlargement of the fields outlined in (D) and (F). Enlargement of autolysosome (H) and autophagosome (I) from (G). Scale bar, 2 μm (for D, F); 1 μm (for E, G); 200 nm (for H, I).
J. LpqH induces colocalization of autophagosomes (endogenous LC3, red) and lysosomes (LAMP-1, green) in human monocytes. Pre-treatment of bafilomycin A1 (Baf-A; 100 nM, 2 h) significantly reduces the extent of colocalization of autophagosomes and lysosomes (right). Representative immunofluorescence images of three representative experiments are shown. The bottom panels are enlargement of the corresponding fields outlined in the top panel. *P ≤ 0.01, versus control conditions. Scale bars, 20 μm (top); 5 μm (bottom). Right, the graph shows the number of profiles in the fields.
K. Immunoblot analyses with Abs to LC3 or β-actin for human monocytes. The cells were stimulated with LpqH in the absence or presence of bafilomycin A1 (Baf-A; 200 nM, 2 h) or chloroquine (Chq; 10 μM, 30 min). Representative gel images of three independent replicates are shown. The ratio of the intensities of the LC3-II and LC3-I bands is indicated below each lane.
U, untreated conditions containing 10% of human sera without LpqH. Rapa, rapamycin-treated; SC, treated with solvent control (0.1% DMSO, for E).

A. Human monocytes were incubated with LpqH (100 ng ml⁻¹) for 12 h; results (means ± SD) are normalized to β-actin expression and are presented relative to expression in LpqH-stimulated cells. *P < 0.0001, versus control conditions.
B and C. Human primary monocytes transduced with lentiviruses expressing non-specific shRNA (shNS), specific shRNAs for LL-37 (shLL-37) or DEFB4 (shDEFB4) were incubated in the absence or presence of LpqH (100 ng ml⁻¹) for 24 h, and subjected to confocal analysis, as described in Fig. 1A. Representative images (three independent experiments) are shown (for B); top, RT-PCR for transfection efficiency. Data shown (for B and C, bottom) represent the means ± SD of three independent samples, with each experiment including at least 250 cells scored in five random fields. *P < 0.001, versus control conditions. Scale bar, 10 μm.
D and E. Human primary monocytes were transduced with shLL-37 (for D), shBeclin-1, shAtg5 (for E) or shNS for 48 h. For (D), cells were then pre-treated with SC, 3-MA (10 μM, 2 h) or Wortmannin (WM; 100 nM, 45 min) before the addition of Mtb (moi = 1). After infection, the cells were treated with LpqH (100 ng ml⁻¹) for 2 or 4 days, and then subjected to the cfu assay; inset, RT-PCR for transfection efficiency.

A and C. Immunoblot analyses with Abs to p-AMPKα or t-AMPKα for human monocytes. Human primary monocytes were incubated with LpqH (100 ng ml⁻¹) for indicated times (for A). Human monocytes were pre-treated with or without BAPTA-AM (5, 10, 25 μM; for C).
B. THP-1 cells were loaded with Fluo-2/AM and stimulated with LpqH, then imaged by Leica TCS SP2 confocal microscopy under a 10/0.40 CS objective lens at 5 s intervals. Top, representative images are shown (at least three independent experiments); bottom, quantification of data. Bottom data show the changes in mean fluorescence intensity from 15 cells per microscopic field over time.
D. Human THP-1 cells transfected with non-specific siRNA (siNS) or specific siRNA for hAMPK (siAMPK) or hCaMKK (siCaMKII) were incubated in the absence or presence of LpqH (100 ng ml⁻¹) for 24 h, and subjected to confocal analysis as described in Fig. 1A. Graph is shown percentage of LC3 punctated cells; inset, RT-PCR for transfection efficiency. Data shown represent the means ± SD of three independent samples, with each experiment including at least 200 cells scored in five random fields.
E and F. Human THP-1 cells were loaded with Fluo-2/AM and stimulated with LpqH in the absence or presence of Src inhibitors PP1, PP2 or analogue PP3 (10 μM; for E) or PLC inhibitor U73122 or analogue U73343 (5 μM; for F), then analysed using live cell imaging by time-lapse microscope (Nikon Eclipse TE2000).
#P < 0.01; *P < 0.002, versus control conditions. Scales bars, 50 μm. U, untreated and incubated; SC, treated with solvent control (0.1% DMSO, for C).

A schematic model for molecular mechanisms of LpqH-induced antibacterial autophagy against mycobacteria. Engagement of TLR2/1 and CD14 results in a rapid phosphorylation of Src kinase at Tyr416, which leads to intracellular Ca²⁺ influx through PLC-γ activation. Increased Ca²⁺ release induces a phosphorylation of AMPKα at Thr172, leading to a downstream activation of p38 MAPK. Activation of intracellular signalling pathways can contribute to upregulate Cyp27b1 hydroxylase induction, which can convert the vitamin D prohormone 25D3 into 1,25D3. The ligation of VDR leads to its increased translocation into nucleus and hormone-dependent transcriptional activation (Liu et al., 2009), resulting in an upregulation of antimicrobial peptide cathelicidin. As in case of 1,25D3-dependent autophagy (Yuk et al., 2009), cathelicidin plays an essential role for antibacterial autophagy, resulting in a fusion of autophagosomes with lysosomes, and finally eliminates intracellular mycobacteria.