NF-kappaB activation during Rickettsia rickettsii infection of endothelial cells involves the activation of catalytic IkappaB kinases IKKalpha and IKKbeta and phosphorylation-proteolysis of the inhibitor protein IkappaBalpha

Abstract

Rocky Mountain spotted fever, a systemic tick-borne illness caused by the obligate intracellular bacterium Rickettsia rickettsii, is associated with widespread infection of the vascular endothelium. R. rickettsii infection induces a biphasic pattern of the nuclear factor-kappaB (NF-kappaB) activation in cultured human endothelial cells (ECs), characterized by an early transient phase at 3 h and a late sustained phase evident at 18 to 24 h. To elucidate the underlying mechanisms, we investigated the expression of NF-kappaB subunits, p65 and p50, and IkappaB proteins, IkappaBalpha and IkappaBbeta. The transcript and protein levels of p50, p65, and IkappaBbeta remained relatively unchanged during the course of infection, but Ser-32 phosphorylation of IkappaBalpha at 3 h was significantly increased over the basal level in uninfected cells concomitant with a significant increase in the expression of IkappaBalpha mRNA. The level of IkappaBalpha mRNA gradually returned toward baseline, whereas that of total IkappaBalpha protein remained lower than the corresponding controls. The activities of IKKalpha and IKKbeta, the catalytic subunits of IkappaB kinase (IKK) complex, as measured by in vitro kinase assays with immunoprecipitates from uninfected and R. rickettsii-infected ECs, revealed significant increases at 2 h after infection. The activation of IKK and early phase of NF-kappaB response were inhibited by heat treatment and completely abolished by formalin fixation of rickettsiae. The IKK inhibitors parthenolide and aspirin blocked the activities of infection-induced IKKalpha and IKKbeta, leading to attenuation of nuclear translocation of NF-kappaB. Also, increased activity of IKKalpha was evident later during the infection, coinciding with the late phase of NF-kappaB activation. Thus, activation of catalytic components of the IKK complex represents an important upstream signaling event in the pathway for R. rickettsii-induced NF-kappaB activation. Since NF-kappaB is a critical regulator of inflammatory genes and prevents host cell death during infection via antiapoptotic functions, selective inhibition of IKK may provide a potential target for enhanced clearance of rickettsiae and an effective strategy to reduce inflammatory damage to the host during rickettsial infections.

FIG. 1.

Analysis of steady-state p65 and p50 mRNA and protein levels during R. rickettsii infection. (A) Uninfected ECs (Con) or cells infected with R. rickettsii for the indicated times were processed for the isolation of total RNA. A total of 20μg of RNA for each experimental condition was subjected to Northern blot analysis with p65 and p105/p50 probes. A representative blot for each probe is shown. (B) Equal volumes of total protein lysates prepared from uninfected (Con) or infected (Rr) ECs at different times were resolved by SDS-PAGE electrophoresis and blotted onto nitrocellulose membrane. The blots were probed with a polyclonal anti-p50 antibody, followed by a monoclonal anti-α-tubulin to control for sample loading.

FIG. 2.

IκBα mRNA expression during R. rickettsii infection. (A) ECs were either left untreated or infected with R. rickettsii for the indicated times prior to isolation of total RNA. A total of 20 μg of RNA for each condition was subjected to Northern blot analysis with an IκBα probe, followed by analysis with a probe for the housekeeping gene GAPDH. A representative blot is shown. (B) Northern blots (n = 4) were scanned, and band intensities were quantified as described in the text. Values for IκBα were first normalized to those for GAPDH, and infected conditions were then compared to the corresponding untreated condition at each time. Changes were averaged across all experiments and are presented as the fold change relative to untreated conditions (mean ± the SE).

FIG. 3.

Western blot analysis of steady-state IκBα and phospho-IκBα (Ser-32) protein levels during R. rickettsii infection. (A) Cultures were infected with R. rickettsii (Rr) or left uninfected (Con) for the indicated number of hours prior to preparation of cellular protein extracts. Equal volumes of samples were separated by SDS-PAGE. Blots were sequentially probed with antibodies against phospho-IκBα, total IκBα, and α-tubulin. A representative blot is shown. Extracts from either untreated or TNF-α-treated HeLa cells (New England Biolabs) were used as positive controls to identify the appropriate total IκBα and phospho-IκBα bands, respectively (not shown). (B) Western blots were scanned and band intensities were quantified as described in the text. Phospho-IκBα (n = 4) and total IκBα (n = 3) values were first normalized to the α-tubulin values and then compared to values obtained from untreated conditions. Changes were averaged across all experiments and are presented as the fold change relative to untreated conditions (mean ± the SE). Error bars for total IκBα fall near or within the closed circles used for the datum points.

FIG. 4.

Changes in IKKα activity during R. rickettsii infection. (A) Assay for quantitative measurement of IKK activity. ECs were incubated with TNF-α (20 ng/ml for 10 min), LPS (E. coli O111:B4; 10 μg/ml for 60 min), or culture medium alone (Con). For each sample, whole-cell extract (20 μg of protein) was subjected to immunoprecipitation with 2 μg each of IKKα-specific antibody (lane 1), normal mouse IgG (lane 2, negative control for specificity), or no antibody (lane 3, control for background phosphorylation). The kinase activity of immunoprecipitates was determined by in vitro phosphorylation of GST-IκBα substrate. The results of a typical IP-kinase assay are shown. (B) R. rickettsii infection activates IKK activity. ECs were infected with rickettsiae for different times corresponding to early and late phases of NF-κB activation or stimulated with TNF-α (positive control). IKKα activity was measured by IP-kinase assay with GST-IκBα(1-54) substrate. (C) Kinetics of Rickettsia-induced IKKα activity. The experiments were performed as described in panel B, and the activity of IKKα was analyzed at selected time points during the course of infection. Representative films from independent observations were scanned and quantified densitometrically. In each experiment, the basal activity in uninfected cells was determined by averaging the band intensity units at different times. The induction above the baseline value was then calculated. The results are presented as the mean ± the SE (n ≥ 3).

FIG. 5.

Effect of R. rickettsii infection on IKKβ activity. (A) Activation of endogenous IKKβ. EC were infected with R. rickettsii organisms for different times corresponding to early and late phases of NF-κB activation or stimulated with TNF-α. IKKβ activity was measured by IP-kinase assay with GST-IκBα(1-54) substrate. (B) Kinetics of Rickettsia-induced IKKβ activity. The activity of IKKβ was analyzed by using a separate set of aliquots of cellular lysates described above for Fig. 4.

FIG. 6.

Inactivation of Rickettsia inhibits infection-induced IKK activity and nuclear translocation of NF-κB. Rickettsia organisms were inactivated by heat treatment (65°C for 30 min.) or formaldehyde fixation (3.7% [vol/vol] for 30 min at 25°C). ECs were incubated with viable (Rr), heat-inactivated (Rr Heated), or formalin-fixed (Rr FF) rickettsiae for 2 h, followed by the isolation of total protein extracts. The results for the activities of IKKα and IKKβ are shown in panels A and B. For comparison, the basal levels of activities in uninfected ECs (Con) were assigned a value of 1. The data presented are the mean ± the SE from three independent experiments. The symbols “✽” and “#” indicate significant changes (P ≤ 0.05) in relation to Con and Rr, respectively. (C) Results of a typical gel retardation assay to investigate activation of NF-κB. Nuclear proteins (2.5 μg per condition) were incubated with ³²P-labeled consensus NF-κB oligonucleotide to measure the extent of DNA-protein binding. The specificity of the binding reactions was ensured by including a 100-fold excess of unlabeled probe (+cold).

FIG. 7.

Effects of IKK inhibitors on R. rickettsii-mediated IKK and NF-κB activation. (A) Parthenolide effectively blocks infection-induced IKKα activity. After preincubation with medium alone (Con) or with medium containing aspirin (+ASP) and parthenolide (+PAR) for 30 min, ECs were left uninfected, exposed to TNF-α, or infected with R. rickettsii for 2 h. IKKα activity is presented as the fold induction relative to the basal level in untreated, uninfected cells (Con). The values are the mean ± the SE from three independent experiments with the exception of TNF+ASP (n = 2). The P values for different experimental conditions relative to unstimulated, infected, and TNF-α-induced cells are also shown. (B) Aspirin inhibits Rickettsia-induced IKKβ kinase activity. The activity of IKKβ was measured after infection in the presence or absence of aspirin and parthenolide. (C) Effect of specific IKK inhibitor parthenolide (PAR) on R. rickettsii-induced NF-κB activation. Nuclear protein extracts from uninfected control (Con) and ECs infected in the absence (Rr) or presence of PAR (Rr+PAR) for 3 h were subjected to EMSA. The positions of gel-shifted complex for NF-κB, nonspecific complex (NS), and excess free probe are indicated. The specificity of the complex formation was confirmed by incubation with a 100-fold molar excess of unlabeled probe during DNA-protein binding (+cold). Similar results were obtained with aspirin treatment.