Figure 1.
Total cholesterol level is increased in A. phagocytophilum–infected HL-60 cells.
A. phagocytophilum–infected HL-60 cells were harvested on 0, 1, 2 and 3 day p.i., and total cellular cholesterol levels and bacterial burden were determined. Cholesterol concentrations are expressed as micrograms of total cholesterol (esterified and unesterified cholesterol) per milligram of total protein (black triangles). Bacterial burdens are expressed as bacterial numbers per 100 cells (black bars). Data are expressed as mean±standard deviation (n = 3) and are representative of at least three independent experiments with similar results. *, p<0.05 (unpaired two-tailed t test); **, p<0.01 (unpaired two-tailed t-test).
Figure 2.
Free cholesterol is enriched in A. phagocytophilum inclusions.
A. phagocytophilum–infected HL-60 cells (A) and A. phagocytophilum inclusions released from host cells (B) were fixed at 2 d p.i., stained with mouse anti-A. phagocytophilum (green) or normal mouse IgG and filipin (blue), and analyzed by fluorescence microscopy. The experiment shown is representative of at least three independent experiments. Bar, 5 µm. Ap, A. phagocytophilum.
Figure 3.
A. phagocytophilum infection requires cholesterol derived from the host LDL uptake pathway rather than de novo biosynthesis.
(A) A. phagocytophilum growth in HL-60 cells cultured in the growth medium containing lipoprotein deficient serum (LPDS), LPDS reconstituted with lipoproteins (LP), and fetal bovine serum were determined on days 1 and 2 p.i. (B) HL-60 cells were pre-treated with anti-LDLR mAb and isotype control mouse IgG and infected with host cell–free A. phagocytophilum. The number of bacteria was determined on days 3 and 4 p.i. (C) Cholesterol transport and biosynthesis inhibitors U18886A, imipramine, 25-hydroxycholesterol (25-HC), and lovastatin were added at 1 h p.i. at different dosages, and numbers of bacteria were determined on day 2 p.i. Data are expressed as mean±standard deviation (n = 3) and are representative of two independent experiments with similar results. *, p<0.05 (unpaired two-tailed t test); **, p<0.01 (unpaired two-tailed t-test). (D) A. phagocytophilum-infected HL-60 cells at 1 h p.i. were treated with lovastatin (1 µM), U18886A (1 µM), imipramine (20 µM), and DMSO vehicle control, respectively. On day 1 p.i., infected cells were harvested and observed by light microscopy following Diff-Quik staining. Arrows indicate A. phagocytophilum inclusions. Note much smaller sizes of inclusions that contain much fewer numbers of bacteria in U18886A- or imipramine-treated cells than those in untreated or lovastatin-treated cells. N, nucleus. All figures are shown at the same magnification. Bar, 5 µm. (E) Cultures were treated as in (C), and numbers of bacteria were determined on days 1 and 2 p.i. *, p<0.05 (unpaired two-tailed t test); **, p<0.01 (unpaired two-tailed t-test). (F) Lovastatin (1 µM), U18886A (5 µM), imipramine (100 µM), and vehicle control were added to A. phagocytophilum-infected HL-60 on day 1 p.i. (∼40% infected cells), and the number of bacteria was determined on day 2 p.i. Data are expressed as mean±standard deviation (n = 3) and are representative of three independent experiments with similar results. *, p<0.05 (unpaired two-tailed t test); **, p<0.01 (unpaired two-tailed t-test).
Figure 4.
DiI-LDL uptake is enhanced upon A. phagocytophilum infection in HL-60 cells.
Uninfected and A. phagocytophilum–infected HL-60 cells were incubated with increasing concentrations of DiI-LDL at 37°C for 2 h. After DiI-LDL uptake, HL-60 cells were directly observed by fluorescence microscopy without fixation (A), or after being fixed and labeled with anti–A. phagocytophilum (B). Arrows indicate A. phagocytophilum inclusions. Ap, A. phagocytophilum. The amount of cell-associated DiI was determined by fluorometric assay. Data were normalized to total cellular protein, and specific association was determined by subtracting the fluorescence measured in the presence of a 30-fold excess concentration of unlabeled LDL from normalized cell-associated DiI fluorescence (C). Data are expressed as mean±standard deviation (n = 3) and are representative of two independent experiments. *, p<0.05 (unpaired two-tailed t test); **, p<0.01 (unpaired two-tailed t-test).
Figure 5.
LDLR is up-regulated in A. phagocytophilum–infected HL-60 at transcriptional and protein levels.
(A) At day 2 p.i., total RNA was extracted from uninfected and A. phagocytophilum–infected HL-60 cells. Analysis of mRNA amount was performed using quantitative RT-PCR with specific primers for each gene. Transcript levels were normalized to mRNA level of TATA-box binding protein or G3PDH in each sample. Ap, A. phagocytophilum. (B) Cell samples were collected at the indicated time points, membrane fractions were isolated, and western blotting was performed to determine LDLR protein levels in uninfected HL-60 and A. phagocytophilum–infected HL-60 cells. (C) Oxytetracycline (OTC, 10 µg/ml) was added to A. phagocytophilum–infected HL-60 cells at 1 h p.i., RNA was extracted from infected HL-60 cells on days 1 and 2 p.i., and LDLR mRNA was analyzed by quantitative RT-PCR. Transcript levels were normalized to mRNA level of TATA-box binding protein in each sample. Inset shows western blotting result using mAb 5C11 against A. phagocytophilum major surface protein P44. (D) Relative LDLR mRNA in lovastatin-treated cell cultures on day 2 p.i. was analyzed by quantitative RT-PCR with primers specific for LDLR gene. (E) LDLR protein levels in lovastatin- or mock (DMSO)-treated infected cells at day 2 p.i. were analyzed by western blotting using anti-LDLR mAb. Data are expressed as mean±standard deviation (n = 3) and are representative of two independent experiments. *, p<0.05 (unpaired two-tailed t test); **, p<0.01 (unpaired two-tailed t-test).
Figure 6.
SREBP activation is not up-regulated in Anaplasma phagocytophilum–infected HL-60 cells.
Uninfected and A. phagocytophilum–infected HL-60 cells were collected at the indicated time points, and western blotting was performed using anti-SREBP-2 mAb. α-Tubulin was used as the protein input control to normalize each sample. Positive control was set up by incubating uninfected cells with LPDS-conditioned medium overnight. Ratio of band intensities of mature form to precursor form of SREBP was calculated to show the SREBP-2 cleavage. Data are representative of three independent experiments. Ap, A. phagocytophilum. P, precursor form of SREBP; M, mature form of SREBP.
Figure 7.
LDLR mRNA is stabilized in A. phagocytophilum–infected host cells.
Actinomycin D (5 µg/ml) was added to uninfected and A. phagocytophilum–infected HL-60 cells for different periods. Total RNA was isolated, and LDLR mRNA (A) and HMG-CoA reductase mRNA (B) were analyzed by quantitative RT-PCR. Data are expressed as mean±standard deviation (n = 3) and are representative of two independent experiments. The decay rates of LDLR mRNA (A) are significantly different (p<0.01), as tested by two-way ANOVA. (C) Chimeric pLuc/LDLR 3′UTR-2 was transfected into RF/6A cells using FuGene HD reagent, and host cell–free A. phagocytophilum was added to transfected RF/6A cells at 1 day post transfection. Luc mRNA was analyzed on day 2 p.i. by quantitative RT-PCR. Cell samples were normalized by the antibiotic gene zeocin mRNA level. Data are representative of two independent experiments. Ap, A. phagocytophilum. ND, not detectable. **, p<0.01 (unpaired two-tailed t-test).
Figure 8.
ERK signaling pathway is involved in the LDLR up-regulation upon A. phagocytophilum infection.
(A) Western blot analysis was performed using antibodies specific to either the phosphorylated or total ERK1/2. Uninfected and A. phagocytophilum–infected HL-60 cells with or without 10 µM ERK inhibitor U0126 treatment were collected at the indicated time points. α-Tubulin was used as the protein loading control to normalize each sample. The values under the bands show the ratios of band intensities of p-ERK and ERK. Ap, A. phagocytophilum. (B) Bacterial numbers per 100 cells treated with the indicated concentrations of U0126 were determined on days 1 and 2 p.i. (C) Western blot analysis of A. phagocytophilum–infected HL-60 cells on day 2 p.i. treated with the indicated concentrations of U0126 was performed using antibodies specific to phosphorylated or total ERK1/2, or A. phagocytophilum outer membrane protein P44. α-Tubulin was used as the protein input control to normalize each sample. The values under the bands show the ratios of band intensities of p-ERK and P44, normalized to total ERK or α-tubulin, respectively. Data are representative of three independent experiments. (D) HL-60 cells were transfected with control (Ctl) or double-stranded siRNA specific targeting the genes encoding MEK1 and MEK2 (MEK1+2) (3 µg/2×106 cells) using the Amaxa Nucleofection system. Two days after transfection, host cell-free A. phagocytophilum was added to the cells and incubated for additional 2 days. One aliquot of samples were lysed and subjected to Western blotting using antibodies against MEK1/2, ERK1/2, phospho-ERK1/2, and A. phagocytophilum P44 protein. The relative protein amount of MEK1/2, phospho-ERK1/2, and P44 were determined using total ERK1/2 as loading control by densitometry analysis. The values under the bands show the relative ratios of band intensities, with the ratios of those from samples nucleofected with control siRNA arbitrarily set as 1. Results are representative of three independent experiments. (E) LDLR expression in A. phagocytophilum–infected HL-60 cells on day 2 p.i. treated with the indicated concentrations of U0126 was determined by quantitative RT-PCR. Data are expressed as mean±standard deviation (n = 3) and are representative of two independent experiments. *, p<0.05 (unpaired two-tailed t test); **, p<0.01 (unpaired two-tailed t-test).