Effects of Anaplasma phagocytophilum on host cell ferritin mRNA and protein levels

Abstract

Ferritin is a major intracellular iron storage protein and also functions as a cytoprotectant by sequestering iron to minimize the formation of reactive oxygen species. Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis, is an obligate intracellular bacterium that colonizes neutrophils. We have previously reported that human promyelocytic HL-60 cells infected with A. phagocytophilum demonstrate increased transcription of ferritin heavy chain and also that the bacterium stimulates neutrophil NADPH oxidase assembly and degranulation during the initial hours of infection (J. A. Carlyon, W. T. Chan, J. Galan, D. Roos, and E. Fikrig, J. Immunol. 169:7009-7018, 2002, and J. A. Carlyon, D. Abdel-Latif, M. Pypaert, P. Lacy, and E. Fikrig, Infect. Immun. 72:4772-4783, 2004). In this study, we assessed ferritin mRNA and protein levels during A. phagocytophilum infection in vitro using HL-60 cells and neutrophils and in vivo using neutrophils from infected mice. The addition of A. phagocytophilum, as well as Escherichia coli and serum-opsonized zymosan, to neutrophils results in a pronounced increase in ferritin light-chain transcription and a concomitant rise in ferritin protein levels. Neutrophils from A. phagocytophilum-infected mice demonstrate elevated ferritin heavy-chain mRNA expression, a phenomenon consistent with infections by intracellular pathogens. Notably, ferritin protein levels of infected HL-60 cells were markedly diminished in a dose- and time-dependent manner. These studies provide insight into the effects A. phagocytophilum has on the ferritin levels of its host cell.

FIG. 1.

Ferritin mRNA expression in A. phagocytophilum-infected HL-60 cells. Host-cell-free A. phagocytophilum organisms were added to HL-60 cells, and infection was allowed to proceed for 0, 0.5, 1, 3, 6, 12, 24, 48, or 96 h. Uninfected HL-60 cells served as controls. At the appropriate time postinfection, total RNA was isolated from 5 × 10⁶ infected HL-60 cells and used as template for cDNA synthesis. Quantitative PCR was performed using the cDNA templates to assess the relative expression levels of fhc and flc. Data are presented as the mean copies of either fhc or flc transcript per 10³ β-actin transcript copies. Samples were analyzed in triplicate. The error bars indicate standard deviations. The mean results for uninfected and infected cells were compared per time point using the Student t test. Values are statistically significant (*, P < 0.05; **, P < 0.01; and ***, P < 0.001). The results are representative of three independent experiments.

FIG.2.

Ferritin protein levels in HL-60 cells decrease in a time- and dose-dependent manner following A. phagocytophilum infection. Host-cell-free A. phagocytophilum (Ap) organisms were added to 5 × 10⁵ HL-60 cells at ratios of 0.4, 1.1, 3.3, and 10 per cell. Uninfected HL-60 cells served as controls. Four days postinfection, (A) whole-cell lysates (10 μg) were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western immunoblot analysis using antibodies directed against A. phagocytophilum P44 and actin, and (B) whole-cell lysates (30 μg) were assayed for ferritin content by ELISA. Bovine serum albumin (BSA; 30 μg) served as a negative control. Statistical significance was determined using one-way ANOVA followed by Tukey's test. (C) Dose-dependent infection time course. A. phagocytophilum was added to 5 × 10⁵ HL-60 cells at approximate ratios of 0 (white bars), 1 (gray bars), and 10 (black bars) per cell. Whole-cell lysates (30 μg) were assessed for ferritin content at 24, 48, and 96 h postinfection. Statistical significance was determined using two-way ANOVA followed by Bonferroni's test. The mean values indicated by different letters are significantly different. (D) Host-cell-free A. phagocytophilum organisms were added to HL-60 cells at a ratio of approximately 10 organisms per cell, and infection was allowed to proceed for 0, 0.25, 0.5, 1, 2, 5.5, 8, 12, 24, 48, 72, or 96 h. Uninfected HL-60 cells served as controls. Whole-cell lysates (30 μg) generated from 5 × 10⁶ cells per time point were assayed for ferritin content. The means ± standard errors of duplicate samples are presented. Statistical significance (*, P < 0.05; **, P < 0.01) was determined using two-way ANOVA followed by Bonferroni's test. Results are representative of four to six independent experiments. Ctrl, control.

FIG. 3.

Ferritin mRNA expression in A. phagocytophilum-infected neutrophils. (A) RT-PCR analyses. Neutrophils (5 × 10⁵) were incubated in the presence and absence of host-cell-free A. phagocytophilum (Ap; 10 per cell) for 1, 3, or 6 h. At the appropriate time point, total RNA was isolated and RT-PCR was performed targeting human fhc and flc as well as β-actin and A. phagocytophilum 16S rRNA genes and p44 transcripts. Results are representative of three experiments. (B) Quantitative RT-PCR analyses. Neutrophils (5 × 10⁵) were incubated with host-cell-free A. phagocytophilum (10 per cell), E. coli (Ec; 10 per cell), or OpZ (10 per cell) for 1, 3, or 6 h. Total RNA was isolated and converted to cDNA, which was used as template for real-time PCR to quantify the relative expression levels of fhc and flc. Samples were analyzed in triplicate. Data are presented as the mean copies of either fhc or flc transcript per 10³ β-actin transcript copies. Error bars indicate standard deviations. Statistical significance was determined using one-way ANOVA followed by Tukey's test. The mean values indicated by different letters are significantly different. Results are representative of four independent experiments. ctrl, control.

FIG. 4.

Ferritin protein levels in neutrophils following A. phagocytophilum infection. Neutrophils (5 × 10⁵) were incubated with A. phagocytophilum (Ap; 20 per cell), E. coli (20 per cell), or OpZ (20 per cell) for 1, 3, or 6 h. Uninfected cells served as controls. Whole-cell lysates (30 μg) were analyzed for ferritin content. The means ± standard errors of duplicate samples are presented. Statistical significance was determined using two-way ANOVA followed by Bonferroni's test. The mean values indicated by different letters are significantly different. Results are representative of four independent experiments.

FIG. 5.

Ferritin mRNA expression in neutrophils recovered from A. phagocytophilum-infected mice. C3H/HeJ mice were inoculated with A. phagocytophilum (Ap). Sham-inoculated mice served as controls. On days 2 and 8, the mice were sacrificed and Gr-1-positive and -negative cells were isolated. Total RNA was extracted and used as template for RT-PCR analyses targeting murine fhc and flc and A. phagocytophilum p44. Samples were normalized according to murine hypoxanthine phosphoribosyltransferase (HPRT) transcript levels. Transcript levels for fhc, flc, and the HPRT gene for Gr-1-negative cells on day 2 are similar to those observed for day 8 and thus are not shown. Results are representative for four separate experiments. ctrl, control.