Protein kinase A-mediated inhibition of gamma interferon-induced tyrosine phosphorylation of Janus kinases and latent cytoplasmic transcription factors in human monocytes by Ehrlichia chaffeensis

Abstract

Ehrlichia chaffeensis, an obligatory intracellular bacterium of monocytes or macrophages, is the etiologic agent of human monocytic ehrlichiosis. Our previous study showed that gamma interferon (IFN-gamma) added prior to or at early stage of infection inhibited infection of human monocytes with E. chaffeensis; however, after 24 h of infection, IFN-gamma had no antiehrlichial effect. To test whether ehrlichial infection disrupts Janus kinase (Jak) and signal transducer and activator of transcription (Stat) signaling induced by IFN-gamma, tyrosine phosphorylation of Stat1, Jak1, and Jak2 in E. chaffeensis-infected THP-1 cells was examined by immunoprecipitation followed by immunoblot analysis. Viable E. chaffeensis organisms blocked tyrosine phosphorylation of Stat1, Jak1, and Jak2 in response to IFN-gamma within 30 min of infection. Similar results were obtained with human peripheral blood monocytes infected with E. chaffeensis. Heat or proteinase K treatment but not periodate treatment of E. chaffeensis abrogated the inhibitory effect, suggesting that protein factor(s) of E. chaffeensis is responsible for the inhibition of IFN-gamma-induced tyrosine phosphorylation. Preincubation of E. chaffeensis with the Fab fragment of dog anti-E. chaffeensis immunoglobulin G also abrogated the inhibitory effect. On the other hand, monodansylcadaverine, which does not block binding but blocks internalization of ehrlichiae into macrophages, did not have any influence on the tyrosine phosphorylation. These results indicate that ehrlichial binding to host cells is sufficient to inhibit Stat1 tyrosine phosphorylation induced by IFN-gamma. Protein kinase A (PKA) activity in THP-1 cells increased approximately 25-fold within 30 min of infection with E. chaffeensis. In THP-1 cells pretreated with a PKA inhibitor, Rp isomer of adenosine 3',5'-cyclic phosphorothioate, E. chaffeensis-induced inhibition of Stat1 tyrosine phosphorylation was partially abrogated. These results suggest that E. chaffeensis blocks IFN-gamma-induced tyrosine phosphorylation of Jak and Stat through raising PKA activity in THP-1 cells, which may be an important survival mechanism of ehrlichiae within the host cell.

FIG. 1

Tyrosine phosphorylation of Stat1, Jak1, and Jak2 during IFN-γ stimulation in THP-1 cells. THP-1 cells (5 × 10⁶) were incubated with 50 nM PMA for 18 h, washed twice with prewarmed RPMI 1640 medium, and stimulated with or without IFN-γ (1,000 U/ml) for 10 min. Cells were washed twice with prewarmed PBS and lysed in ice-cold lysis buffer. Cell lysates were immunoprecipitated with antibodies specific for Stat1, Jak1, Jak2, or normal rabbit serum (NS) as described in Materials and Methods. The immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis and subjected to Western blot analysis by using antiphosphotyrosine antibody. Stat1 (91 kDa), Jak1 (130 kDa), and Jak2 (130 kDa) are indicated. Data shown are from one of three independent experiments with similar results.

FIG. 2

Time course of the effect of E. chaffeensis infection on tyrosine phosphorylation of Stat1, Jak1, and Jak2 in response to IFN-γ. THP-1 cells preincubated with PMA for 18 h were treated with host cell-free E. chaffeensis organisms for the indicated periods of time and stimulated with IFN-γ for 10 min. The cell lysates were immunoprecipitated with anti-Stat1, anti-Jak1, or anti-Jak2 antibody, and Western blot analysis was performed with antiphosphotyrosine antibody. Similar results were obtained in two experiments.

FIG. 3

Protein factors of E. chaffeensis are required for the inhibition of tyrosine phosphorylation of Stat1. THP-1 cells preincubated with PMA for 18 h were subjected to different treatment for 30 min followed by IFN-γ stimulation for 10 min. The cell lysates were immunoprecipitated with anti-Stat1 antibody and analyzed by immunoblotting. The upper panel represents the membrane probed with antiphosphotyrosine (α-PTyr), and the lower panel represents the membrane probed with anti-Stat1 (α-Stat1). Lanes: 1, untreated control cells without IFN-γ stimulation; 2 to 8, cells stimulated with IFN-γ; 2, untreated cells; 3, cells treated with viable E. chaffeensis; 4, cells treated with heat-killed E. chaffeensis; 5, cells treated with periodate-treated E. chaffeensis; 6, cells treated with proteinase K-treated E. chaffeensis; 7, cells treated with lysate of E. chaffeensis; 8, cells treated with E. coli. Stat1, nonphosphorylated Stat1; Stat1-P, phosphorylated Stat1 (20). Two separate experiments yielded similar results.

FIG. 4

IFN-γ-induced tyrosine phosphorylation of Stat1 is irreversible by E. chaffeensis infection. THP-1 cells were pretreated with PMA for 18 h and then treated with E. chaffeensis for 30 min followed by IFN-γ stimulation for 10 min (lane 3). THP-1 cells were stimulated with IFN-γ for 10 min and then incubated with E. chaffeensis for 30 min (lane 4). Lanes: 1, untreated control cells without IFN-γ stimulation; 2, untreated cells stimulated with IFN-γ. Similar results were obtained in two experiments. For other details, see the legend to Fig. 3.

FIG. 5

Effects of the Fab fragment of anti-E. chaffeensis antibody and monodansylcadaverine on E. chaffeensis-induced inhibition on Stat1 tyrosine phosphorylation. (A) THP-1 cells pretreated with PMA for 18 h were treated for 30 min with E. chaffeensis preincubated with the Fab fragment of anti-E. chaffeensis antibody or Fab fragment of normal dog IgG. After stimulation with IFN-γ for 10 min, cell lysates were immunoprecipitated with anti-Stat1 antibody and analyzed by immunoblotting. Lanes: 1, untreated control cells without IFN-γ stimulation; 2 to 5, cells stimulated with IFN-γ; 2, untreated cells; 3, cells treated with E. chaffeensis; 4, cells treated with E. chaffeensis preincubated with Fab fragment of anti-E. chaffeensis antibody; 5, cells treated with E. chaffeensis preincubated with Fab fragment of normal dog IgG. (B) THP-1 cells preincubated with PMA for 18 h were treated with E. chaffeensis for 30 min in the absence (lanes 1 to 3) or presence (lanes 4 to 6) of monodansylcadaverine (250 μM). Monodansylcadaverine was added 3 h prior to the addition of E. chaffeensis. The cells were then stimulated with IFN-γ for 10 min. Lanes: 1 and 4, untreated control cells; 2 and 5, untreated cells stimulated with IFN-γ; 3 and 6, cells treated with E. chaffeensis and stimulated with IFN-γ. Similar results were obtained in two independent experiments. For other details, see the legend to Fig. 3.

FIG. 6

Effect of a PKA inhibitor on E. chaffeensis-induced inhibition of Stat1 tyrosine phosphorylation. THP-1 cells preincubated with PMA for 18 h were treated with E. chaffeensis for 30 min in the absence or presence of Rp-cAMP[S] (lanes 4 to 6, 50 μM; lane 7, 100 μM). Rp-cAMP[S] was added 3 h prior to the addition of E. chaffeensis. The cells were then stimulated with IFN-γ for 10 min. Lanes: 1 and 4, untreated control cells without IFN-γ stimulation; 2 and 5, untreated cells stimulated with IFN-γ; 3, 6, and 7, cells treated with E. chaffeensis and stimulated with IFN-γ. Similar data were obtained in two experiments. For other details, see the legend to Fig. 3.

FIG. 7

Effect of E. chaffeensis infection on tyrosine phosphorylation of Stat1, Jak1, and Jak2 in human peripheral blood monocytes. Human peripheral blood monocytes were treated with E. chaffeensis for 30 min followed by IFN-γ stimulation for 10 min. Cell lysates were immunoprecipitated with antibodies for Stat1, Jak1, or Jak2 and subjected to Western blot analysis. Lanes: 1, 4, and 7, untreated control cells without IFN-γ stimulation; 2, 5, and 8, untreated cells stimulated with IFN-γ; 3, 6, and 9, cells treated with E. chaffeensis and stimulated with IFN-γ. The data presented are from one of two independent experiments that gave similar results.