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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-κB (NF-κB) 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-κB subunits, p65 and p50, and IκB proteins, IκBα and IκBβ. The transcript and protein levels of p50, p65, and IκBβ remained relatively unchanged during the course of infection, but Ser-32 phosphorylation of IκBα at 3 h was significantly increased over the basal level in uninfected cells concomitant with a significant increase in the expression of IκBα mRNA. The level of IκBα mRNA gradually returned toward baseline, whereas that of total IκBα protein remained lower than the corresponding controls. The activities of IKKα and IKKβ, the catalytic subunits of IκB 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-κB 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 IKKα and IKKβ, leading to attenuation of nuclear translocation of NF-κB. Also, increased activity of IKKα was evident later during the infection, coinciding with the late phase of NF-κB activation. Thus, activation of catalytic components of the IKK complex represents an important upstream signaling event in the pathway for R. rickettsii-induced NF-κB activation. Since NF-κB 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.
Rocky Mountain spotted fever, caused by the obligate intracellular bacterial pathogen Rickettsia rickettsii, is one of the most frequently reported and severe rickettsial diseases in the United States. R. rickettsii primarily invades vascular endothelial cells (ECs) in humans, replicates predominantly within the cytoplasm, and utilizes actin polymerization-based directional motility for intracellular movements and intercellular spread (19, 42). The vascular endothelium is a multifunctional endocrine and paracrine organ involved in the modulation of blood flow and vessel tone, coagulation, and regulation of immune and inflammatory responses. In general, an intricate relationship between activation of immune responses and modulation of coagulation properties, usually followed by dysregulation of hemostatic mechanisms, is a hallmark feature of infectious diseases affecting the endothelium (2, 45). A majority of pathological sequelae associated with spotted fever group rickettsioses are attributed to damage of ECs affecting these functions in severe cases of infection (41, 42). ECs not only participate in the uptake of viable Rickettsia organisms attached to the cell surface through “induced phagocytosis,” resulting in internalization, but actively respond to infection by adjusting the expression of mediators with important physiological functions such as adhesion molecules, cytokines, chemokines, and regulatory components of the coagulation cascade (8, 40). Many of these genes are regulated by the nuclear factor-κB (NF-κB)/Rel family of transcription factors, known to play an important role in immediate-early pathogenic responses (1, 2, 12).
The prototypical NF-κB complex is a heterodimer of RelA (p65) and p50 (NFKB1) subunits; other members of the Rel family include c-Rel, RelB, and p52 (NFKB2). In resting cells, NF-κB is sequestered in the cytoplasm in its latent form through association with inhibitory proteins termed IκB (IκBα, IκBβ, IκBγ, IκB, p105, and p100). Upon appropriate cell stimulation, IκBα and IκBβ are rapidly phosphorylated on specific amino-terminal serines, signaling for ubiquitination and degradation by the 26S proteasome (12, 46). This results in the exposure of a nuclear localization sequence (NLS) and DNA-binding domains, allowing NF-κB to enter the nucleus and stimulate the transcription of target genes. One such target is IκBα itself, the resynthesis of which completes an autoregulatory loop to ensure that the activation of NF-κB is turned off in a timely manner (12).
Despite sharing many common structural and functional properties (44), IκBα and IκBβ exhibit significant differences in their affinity for NF-κB dimers (35). A recent study further suggests unique injury-context-specific roles for IκBα and IκBβ in NF-κB-mediated cellular responses (9). Some agonists cause a transient activation of NF-κB, e.g., tumor necrosis factor alpha (TNF-α) and phorbol ester, inducing the rapid degradation and resynthesis of IκBα. However, other signals such as lipopolysaccharide (LPS) and interleukin-1 (IL-1) also cause the slower degradation of IκBβ (39). Newly synthesized IκBβ in a hypophosphorylated state is capable of binding to NF-κB without masking the nuclear localization sequence, effectively preventing sequestration and subsequent inactivation of NF-κB by newly synthesized IκBα, resulting in persistent NF-κB activation (38). Thus, these differences are specific to variations in the phosphorylation state of IκBβ in response to a subset of inducers. A critical advance in the understanding of IκB phosphorylation was the discovery of a multisubunit, high-molecular-weight complex termed IκB kinase (IKK), which phosphorylates both IκBα and IκBβ. IKK contains two catalytically active subunits, IKKα and IKKβ (also referred to as IKK-1 and IKK-2), which form homo- and heterodimers through leucine zipper domains that bind to a third protein IKKγ (also called NEMO [for NF-κB essential modulator]). Although IKKγ is a regulatory subunit with no catalytic activity, it is indispensable for coupling the IKK complex to upstream activators (17, 46).
Our laboratory has shown that activation of NF-κB plays a crucial role during R. rickettsii infection of ECs by controlling the expression of several procoagulant and proinflammatory genes (34) and by exerting its antiapoptotic functions to protect the host cells from apoptotic death (3, 22). Interestingly, the kinetics of R. rickettsii-induced NF-κB activation in ECs follows a biphasic pattern characterized by an early transient phase peaking at about 3 h postinfection (hpi) and a second sustained phase occurring between 18 and 24 h (37). In the present study, we describe the expression of the primary NF-κB proteins p65 and p50, the phosphorylation and proteolysis of IκB proteins, and the activation of catalytic components of IKK signalosome during R. rickettsii infection of ECs. The effect of specific IKK inhibitors on R. rickettsii-induced NF-κB activation was also investigated.
All experiments were performed with human umbilical vein ECs cultures established as described previously (3, 37). ECs, seeded at a dilution to achieve 80 to 90% confluence after 3 to 4 days, were routinely used after a second passage in culture. Cultures were infected with the Sheila Smith strain of Rickettsia rickettsii by using a seed stock (1 × 107 to 5 × 107 PFU/ml) prepared from infected Vero cells (37). Unless stated otherwise, EC monolayers were infected with ca. 6 × 104 PFU of viable rickettsia organisms for every square centimeter of culture area. As reported in our earlier studies, this protocol resulted in infection of ca. 75% of total cell population with two to three rickettsiae per cell at 3 h and ≥80% cells with three to four rickettsiae at 6 h (37). For inactivation, aliquots of rickettsial preparations were subjected to either heat treatment or fixation with formaldehyde (3.7% [vol/vol]) (30). Formaldehyde was removed by three washes with K36 buffer (0.1 M KCl, 0.15 M NaCl, 0.05 M potassium phosphate buffer [pH 7.0]) and centrifugation at 10,000 × g for 10 min.
Total RNA was isolated from uninfected and infected ECs at 3, 7, 14, and 21 hpi by using Tri-Reagent (Molecular Research Center, Inc., Cincinnati, Ohio) according to the manufacturer's instructions. Northern blot analysis was performed essentially as described previously (14). Briefly, 20 μg of total RNA per condition was denatured in a glyoxal-dimethyl sulfoxide mix and then separated by electrophoresis in 1.4% agarose gels with 10 mM sodium phosphate buffer (pH 7.0) with recirculation. RNA was transblotted to Zeta-probe membrane (Bio-Rad Laboratories, Hercules, Calif.) in 0.5× TAE buffer (Tris-acetate, EDTA). The membrane was air dried, and RNA was fixed by baking at 80°C in vacuo for 1.5 h. Blots were prehybridized in 0.5 M Na2HPO4 (pH 7.2), 1 mM EDTA, and 7% sodium dodecyl sulfate (SDS) for at least 1 h. For detection of p65 and p105/p50, plasmids containing murine p65 and human p105/p50 cDNA (kindly provided by Edward Schwarz and Albert Baldwin, Jr., respectively) were used as described previously (48, 49). Restriction endonuclease digestion was performed on plasmids containing human IκBα and murine IκBβ (gifts from Edward Schwarz) to obtain the corresponding probes of 1,100 and 960 bp, respectively. Inserts were isolated by agarose gel electrophoresis, followed by purification by using Sephaglas BP kit (Amersham Biosciences, Piscataway, N.J.) according to the manufacturer's instructions. Prior to hybridization, the probes were radioactively labeled with [32P]dCTP (Perkin-Elmer Life and Analytical Sciences, Boston, Mass.) by using the random primers DNA labeling system according to the manufacturer's instructions (Invitrogen, Carlsbad, Calif.). The labeled probes were denatured by boiling, added to the prehybridization buffer, and incubated with the blots overnight. Blots were washed for 1 h in wash solution 1 (40 mM Na2HPO4 [pH 7.2], 1 mM EDTA, 5% SDS) and then three 30-min washes in wash solution 2 (40 mM Na2HPO4 [pH 7.2], 1 mM EDTA, 1% SDS). The temperatures used during hybridization and washing procedures for the murine and human probes were 58 and 65°C, respectively. For detection of signal, blots were exposed to Kodak Biomax MS film. Blots were stripped by repeating the wash solution protocol above except at 95°C. A human glyceraldehyde-3-phosphate dehydrogenase probe (GAPDH; 1,000 bp; provided by P. J. Simpson-Haidaris) was used to control for variations in sample loading and normalization of densitometric data.
At different times postinfection, protein lysates were prepared by scraping cells into 1× Laemmli buffer (62.5 mM Tris-HCl [pH 6.8], 2% [wt/vol] SDS, 10% glycerol, 50 mM dithiothreitol, 0.1% [wt/vol] bromophenol blue) (25) and sonicated them for 10 s on ice to shear DNA. Equal volumes of total lysates were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto nitrocellulose membrane. The primary antibodies for NF-κB proteins were polyclonal rabbit anti-p50 and anti-p65 (Chemicon, Temecula, Calif.). Polyclonal anti-phospho-IκBα specifically detected phosphorylation at Ser-32, whereas a second polyclonal antibody detected total IκBα independent of N-terminal phosphorylation (Cell Signaling Technology, Beverly, Mass.). For detection of IκBβ proteins, an antibody recognizing an amino-terminal sequence common to both IκBβ1 and IκBβ2 isoforms was used (Santa Cruz Biotechnology, Santa Cruz, Calif.). A monoclonal murine anti-tubulin antibody (Amersham Biosciences) was used to normalize for sample loading. Blots were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies, followed by Renaissance chemiluminescence reagent (Perkin-Elmer Life and Analytical Sciences) and subsequently exposed to ECL Hyperfilm (Amersham Biosciences).
For immunoprecipitation-kinase (IP-kinase) assays, cellular protein extracts were prepared in a buffer containing protease and phosphatase inhibitor cocktails (Sigma-Aldrich, St. Louis, Mo.) as described previously (5), and the protein concentration was determined by Bradford assay. Equal amounts of sample protein (20 to 50 μg) were subjected to IP with either anti-IKKα or anti-IKKβ (Santa Cruz Biotechnology) or normal mouse immunoglobulin G (IgG) as a negative control. Typically, 2 μg of IKKα or 3 μg of IKKβ antibody was mixed with the cell lysates and incubated on a rocker platform for at least 2 h at 4°C. Protein A-Sepharose beads (10 μl) were then added, and the samples were incubated for an additional hour with gentle rotation. The protein A-Sepharose immunopellets were sedimented by centrifugation at 2,000 rpm for 2 min and washed twice with the IP buffer (20 mM HEPES-NaOH [pH 7.6], 40 mM sodium β-glycerophosphate, 20 mM sodium fluoride, 1 mM sodium orthovanadate [activated], 20 mM p-nitrophenyl phosphate, 1 mM dithiothreitol, 0.1% [vol/vol] NP-40, 1 mM phenylmethylsulfonyl fluoride, and 10 μg of protease inhibitor cocktail/ml) supplemented with 0.4 M sodium chloride. The immune complexes for IKKα were then washed with IP buffer containing 0.4 M sodium chloride and 2 M urea. After equilibration in a kinase reaction buffer (20 mM HEPES-NaOH [pH 7.6], 20 mM sodium β-glycerophosphate, 10 mM magnesium chloride, 0.1 mM sodium orthovanadate [activated], 10 mM p-nitrophenyl phosphate, 50 mM sodium chloride, 2 mM dithiothreitol, and 10 μg of protease inhibitor cocktail/ml), phosphorylation reactions were performed at 30°C in 20 mM HEPES-NaOH (pH 7.6), supplemented with 20 μM ATP, 5 μCi of[γ-32P]ATP, and 3 μg of glutathione S-transferase (GST)-IκBα(1-54) substrate (Boston Biologicals, Wellesley, Mass.). Specificity of the kinase reaction was confirmed by using mutant GST-IκBα(1-54-AA), in which alanines are substituted for serines 32 and 36 (provided by J. DiDonato). The reactions were terminated after 30 min by heat denaturation in the presence of 1% SDS, and radiolabeled phosphoproteins were resolved by SDS-PAGE. The IgG bands on the gels were quantified to control for possible variations among the amounts of immunocomplex loaded in each lane. The gels were then dried at 80°C for 1 h in vacuo. Bands were detected by exposure to Biomax film (Kodak, Rochester, N.Y.) at −80°C.
Nuclear extracts from ECs were prepared, and the presence of DNA-binding activity was analyzed by using an oligonucleotide probe (22 bp, 5′-AGT TGA GGG GAC TTT CCC AGG C-3′) containing a consensus NF-κB sequence (3, 31). End labeling of the probe was done by using T4 polynucleotide kinase according to the manufacturer's instructions (Promega Corp., Madison, Wis.). Binding reactions for the formation of DNA-protein complexes and subsequent resolution on native polyacrylamide gels (4% [wt/vol]) were performed by using Promega's electrophoretic mobility gel shift assay (EMSA) system (37). Total protein (5 to 8 μg) from extracts prepared for the IP-kinase assay were also used for EMSAs where noted.
The autoradiograms were scanned in the grayscale mode by using a Hewlett-Packard ScanJet scanner at a resolution setting of 300 to 600 dpi. Volume analysis was performed by using ImageQuant software (v. 3.3; Molecular Dynamics, Sunnyvale, Calif.). The results are reported as means ± the standard errors (SE) or standard deviations as noted.
In a previous study, we showed that the major NF-κB/DNA complex activated during R. rickettsii infection is the prototypical heterodimer of p65 and p50 DNA-binding subunits (37). The p105 protein is an inactive precursor, which is processed upon cell stimulation to produce the active p50 subunit with DNA-binding capability. Thus, analysis of p50 also represents a measure of the p105 precursor levels (29). The possibility that altered expression of p105/p50 or p65 might play a role in Rickettsia-induced NF-κB activation has precedent from studies suggesting that infection of cultured fibroblasts by human cytomegalovirus results in the upregulation of the message for both p105 and p65 through modulation by both viral and cellular factors (48, 49). To determine the pattern of mRNA expression of these subunits, we performed Northern hybridization with specific cDNA probes and total RNA isolated from ECs at 3, 7, 14, and 21 hpi. In three independent experiments, no significant differences in the steady-state level of mRNA for p65 were evident, whereas that of p105/p50 exhibited a slight increase over basal expression (Fig. (Fig.1A).1A). Fold differences ranged from (1.23 ± 0.03)- to (1.4 ± 0.2)-fold (mean ± the SE) for p65 and from (1.5 ± 0.3)- to (2.1 ± 0.3)-fold for p105 in R. rickettsii-infected ECs over the 21-h time course compared to uninfected controls. This observation was further confirmed by immunoblot analysis to monitor the protein levels of p65 and p50 in cell lysates obtained from uninfected and infected ECs at various times. In agreement with the mRNA data, no noticeable changes were detected in the steady-state protein levels of p65 (data not shown), and subtle changes in p105/p50 mRNA expression were not reflected by detectable changes in the level of p50 protein (Fig. (Fig.1B1B).
One of the major pathways responsible for the regulation of NF-κB activation involves the phosphorylation and degradation of IκBα and/or IκBβ by the 26S proteasome with subsequent resynthesis of IκBα via an autoregulatory feedback mechanism. Therefore, we sought to determine the kinetics of mRNA expression, as well as the extent of proteolysis of IκBα and IκBβ proteins during R. rickettsii infection. Low steady-state levels of IκBα mRNA were detected in uninfected ECs. After infection, however, the abundance of IκBα mRNA at 3 hpi was increased by (6.6 ± 3.0)-fold in comparison to the uninfected control. This was followed by a decline toward the baseline, but IκBα expression was still higher than the basal level at all times tested (Fig. 2A and B). Due to alternative splicing, the human IκBβ gene gives rise to two mRNA species of ca. 1.8 and 2.8 kb, corresponding to IκBβ1 and IκBβ2 proteins, respectively (20). Keeping this in mind, an IκBβ probe able to hybridize with both mRNA species was used in the present study. Quantitation of Northern blot autoradiographs revealed that both IκBβ transcripts remained relatively unchanged in comparison to uninfected ECs, showing insignificant variations in their expression at the level of transcription (not shown).
The phosphorylation status of both IκBα and IκBβ proteins over the time course was also examined by immunoblotting. Uninfected ECs had negligible or very low levels of phospho-IκBα at all time points measured. Increased phosphorylation of IκBα was clearly evident at 1.5 and 3 hpi in apparent correlation with the initial phase of NF-κB activation. Phosphorylation subsequently declined toward the basal level at later times. In accordance with this finding, significantly lower cellular levels of total IκBα were present at 1.5 and 3 h after infection, indicating proteasomal degradation. Interestingly, steady-state levels of total IκBα protein were consistently low over the entire time course, ranging from (0.38 ± 0.09)- to (0.53 ± 0.04)-fold in comparison to expression in uninfected ECs (Fig. (Fig.3).3). The lower levels of IκBα protein occurred despite evidence for increased transcription of IκBα (Fig. (Fig.2),2), which likely results in new IκBα synthesis. In contrast, little change in the expression of IκBβ1 and IκBβ2 were seen in R. rickettsii-infected ECs at any time during the course of infection (results not shown), with the exception of moderately lower IκBβ1 levels at 18 hpi ([0.65 ± 0.16]-fold in comparison to the uninfected control [n = 4]).
Since IKKα and IKKβ, the catalytic moieties of IKK complex, can directly phosphorylate IκB proteins, we next examined the activation of endothelial IKKα and/or IKKβ to gain further insight into upstream signaling mechanisms responsible for the biphasic pattern of NF-κB induction during R. rickettsii infection. IKKα and IKKβ were immunoprecipitated from total protein lysates of infected ECs, and their activities were determined by in vitro kinase assays with a fusion protein GST-IκBα(1-54). This substrate contains the specific phosphorylation sites Ser-32 and Ser-36 and eliminates the possibility of nonspecific phosphorylation at the C terminus of full-length IκBα protein. Initially, ECs were treated with either TNF-α (20 ng/ml for 10 min) or LPS (10 μg/ml for 1 h), known inducers of IKK activation in other cell types (10), to optimize the conditions for immunoprecipitation. Specificity was ensured by using normal mouse IgG as a negative control, which failed to precipitate IKKα or IKKβ (not shown). Both TNF-α and LPS caused a significant induction of IKK activity in EC (Fig. (Fig.4A).4A). In comparison to the rapid activation within minutes following TNF-α treatment, R. rickettsii infection induced a slower increase in IKK activity (Fig. (Fig.4B4B and and5A).5A). Infection of ECs for 90 min resulted in a significant increase in IKKα activation, which peaked at 120 min (Fig. 4B and C). Moreover, the extent of IKKβ activation at these early times postinfection (30 to 120 min) was similar to that of IKKα (Fig. (Fig.5).5). Thus, maximal activation of IKK occurred at approximately the same time at which IκBα was maximally phosphorylated (Fig. (Fig.3)3) and was consistent with the kinetics of the early transient phase of NF-κB activation (Fig. (Fig.66 and and7).7). Similarly, both IKKα and IKKβ activities were increased later in infection, coinciding with the late, sustained phase of NF-κB activation at 18 to 24 hpi. IKKα activation was comparable to that attained at 2 hpi. i.e., (3.33 ± 0.94)-fold at 24 h (P = 0.09 versus uninfected control). IKKβ activation, however, was found to be more moderate than that observed early in infection, i.e., (1.56 ± 0.40)-fold at 24 h (P = 0.20 against uninfected control).
To identify the factor(s) responsible for NF-κB activation, we took the approach of infecting EC with Rickettsia organisms that had been inactivated by exposure to heat (65°C) or killed by treatment with 3.7% formaldehyde for 30 min. These treatments result in complete inactivation of rickettsiae (30, 43) without interfering with measurements of IKK or DNA-binding activity of NF-κB. Considering that the concentrations of formaldehyde used to inactivate Rickettsia may adversely affect the viability of ECs in culture, such rickettsial preparations were thoroughly washed prior to use. Heat pretreatment of R. rickettsii prior to contact with ECs led to average reductions of 22 and 41% in the IKKα and IKKβ activities, respectively, in comparison to infection with an identical preparation of viable rickettsiae at 2 hpi (Fig. 6A and B). Gel shift analysis also indicated that heat inactivation of rickettsiae caused ≥80% inhibition of both phases of NF-κB activation. Similarly, formaldehyde treatment of rickettsiae completely abolished infection-induced activation of IKKα and IKKβ and the associated increase in NF-κB activation as analyzed by EMSA (Fig. (Fig.6).6). These results suggest the involvement of a heat-sensitive rickettsial protein in triggering the infection-induced responses of ECs.
To further define the role of IKKα and IKKβ in upstream signaling, the effects of two structurally different anti-inflammatory agents on R. rickettsii-mediated NF-κB activation were investigated. Parthenolide, a sesquiterpene lactone compound, predominantly targets the IKK complex (16), and aspirin has been shown to inhibit IKKβ activity (27). To confirm specificity in our culture system and to determine an effective concentration for subsequent infection experiments, ECs were stimulated with TNF-α in the presence or absence of increasing concentrations of parthenolide (5 to 20 μM), and whole-cell extracts were analyzed in parallel for NF-κB DNA binding (not shown) and IKK activation (Fig. (Fig.7A).7A). Both of these responses were inhibited by parthenolide in a dose-dependent manner, with complete blockade at concentrations of 10 μM or higher. The optimum concentration of aspirin to achieve complete inhibition of TNF-α-induced NF-κB and IKK activation was ≥10 mM. Further experiments with R. rickettsii were carried out with aspirin at a final concentration of 10 mM and parthenolide at 10 μM, since these treatments had no significant effect on the extent of infection as assessed by indirect immunofluorescence microscopy. Although incubation with these compounds alone caused a slight induction of IKK activity in ECs, the apparent changes were found to be statistically insignificant (Fig. 7A and B) and did not result in an increased nuclear translocation of NF-κB (Fig. (Fig.7C).7C). Parthenolide treatment had an inhibitory effect on both R. rickettsii-induced IKKα, as well as IKKβ activities, as measured at 2 hpi, although its effect on IKKα was more pronounced. Aspirin was also able to inhibit infection-induced activation of IKKα and IKKβ. Furthermore, inhibitory effects of aspirin and parthenolide on infection-induced IKK activities were paralleled by a significant reduction (>75%) or complete attenuation of NF-κB DNA-binding activity due to R. rickettsii infection (Fig. (Fig.7).7). Together, these results clearly indicate that NF-κB activation due to infection occurs predominantly through upstream signaling mechanisms leading to the activation of IKKα and IKKβ.
Activation of NF-κB plays a critical role in the pathophysiology of R. rickettsii infection by ensuring the survival of infected host cells to allow for the continued growth and replication of intracellular bacteria (3, 22). A particularly intriguing aspect of Rickettsia-induced NF-κB activation is that it occurs in two distinct phases during infection of cultured ECs (37); there is also a possibility that differences may exist in the upstream signaling mechanisms contributing to this biphasic pattern. The goal of the present study was to define changes in the mRNA and protein expression of major NF-κB subunits, degradation of IκB proteins, and activation of catalytic components of the IκB kinase complex, a cytoplasmic signalosome responsible for the phosphorylation of IκBα and/or IκBβ.
Although NF-κB is known to participate in its own regulation by partially regulating the expression of the human p105/p50 gene promoter (48), the steady-state mRNA of p105 and protein levels of p50 in Rickettsia-infected cells remained relatively unaltered. This suggests that de novo transcription via autoregulatory or other transcription factors (e.g., AP1 and SP1) does not take place during the course of infection. It also indicates that infection did not affect the processing of the p105 precursor to the active p50 form. Similar to other cell types, p65 was expressed constitutively, and R. rickettsii infection did not appear to alter its expression profile. That the p65 promoter contains at least three functional sites for the transcription factor SP1 upstream of the initiation site, but none for NF-κB (48, 49), provides further indirect evidence for noninvolvement of SP1-mediated transcriptional events and supports the notion that SP1 is not activated during R. rickettsii infection (L. A. Sporn and S. K. Sahni, unpublished observations).
The IκB family proteins can differentially associate with and regulate the activity of various NF-κB dimers in the cytoplasm. The targeted degradation of these proteins is critical to all known mechanisms of NF-κB activation. In R. rickettsii-infected ECs, the early peak of NF-κB DNA-binding activity coincided with increased IκBα mRNA levels and was preceded by increased phosphorylation of IκBα (Fig. (Fig.22 and and3).3). Lower levels of total protein further indicate concurrent degradation of phosphorylated IκBα (Fig. (Fig.3).3). This is in agreement with our earlier findings that inhibition of proteasomal IκBα degradation via MG132 or MG115 or expression of a dominant-negative IκBα can effectively inhibit the early phase of Rickettsia-induced NF-κB in ECs or fibroblasts, respectively (3, 31). The failure to accumulate IκBα back to basal levels at all times despite evidence for increased mRNA expression suggests the involvement of continued IκBα degradation, at least in part, in the late phase of NF-κB activation. It is possible that other unknown mechanisms governing posttranscriptional modifications to either inhibit or downregulate the resynthesis of IκBα, which are known to occur during cytomegalovirus infection of monocytes (50) and respiratory syncytial virus infection of A549 cells (21), may also be involved.
In spite of significant sequence homology and ability to interact with the same set of NF-κB proteins, IκBα and IκBβ display distinct responses to different inducers. Persistent NF-κB activation by soluble agonists (e.g., LPS and IL-1), infection with pathogenic bacteria (Listeria), and pathogen-derived mediators (Tax protein of human T-cell leukemia virus type 1) usually involve prolonged activation of NF-κB by binding to hypophosphorylated IκBβ or inactivation of IκBβ (15, 28, 39). We observed only subtle changes in the mRNA and protein expression levels of IκBβ at all times tested in the present study, suggesting that it does not contribute appreciably to NF-κB activation during R. rickettsii infection. IκB, another member of the IκB family, associates predominantly with p65 homodimers or p65-cRel heterodimers in the cytoplasm but not with p50 containing heterodimers. It has been proposed that specific interactions of IκB with c-Rel dimers play a functional role in the induction of expression of adhesion molecules in vascular ECs (36), and degradation of both IκBα and IκB to various degrees occurs during infection of different intestinal ECs with enteroinvasive bacteria (7). Although R. rickettsii infection of ECs triggers nuclear translocation of p65 without affecting c-Rel (37), a possibility which remains to be investigated is that the degradation of IκB associated with p65 (RelA) homodimers may contribute to the second phase of NF-κB activation during rickettsial infection.
Recent studies have established the importance of IKK complex phosphorylation in the canonical pathway of NF-κB activation in response to a variety of factors, including cytokines, pathogenic bacteria, and viruses (2, 33, 46). Increased kinase activity of both IKKα and IKKβ as a consequence of R. rickettsii infection clearly demonstrates modulation of the IKK complex in ECs. Early during the infection, the levels of activation for both kinases were strikingly similar and coincided with the Ser32 phosphorylation of IκBα, an essential signaling step preceding nuclear translocation of NF-κB. Thus, it appears that the early transient phase of Rickettsia-induced NF-κB activation is mediated by mechanisms involving both IKKα and IKKβ. IKK activation at later times apparently corresponded with the second phase of NF-κB, but only IKKα activity achieved levels comparable to those seen early during the infection. The role of IKKα in the late sustained phase of NF-κB activation, however, is unique in that existing biochemical and genetic evidence indicates that, whereas IKKβ serves as the dominant kinase, IKKα performs a potentially redundant function in stimulus-induced NF-κB activation (4). It is also possible that NIK (for NF-κB inducing kinase) or some other upstream mitogen-activated protein kinase(s) may be activated, similar to those stimulated by cytokines. Chlamydia pneumoniae infection of monocytic cells also involves increased activity of IKKα and IκBα/IκB proteolysis (6), but the activation of IKKα in this case is transient and does not resemble the pattern seen in our studies. It should be noted that intracellular R. rickettsii organisms reside and replicate mainly in the host cell cytosol (8, 42), and studies using a “cell-free” system have documented the ability of viable R. rickettsii to directly interact with and enhance the DNA-binding activity of NF-κB, which otherwise remains inactive in cytoplasmic extracts isolated from ECs (31). Such activation of NF-κB occurs in a proteasome-independent fashion and requires a rickettsial protease activity (31, 32). Thus, it is possible that the second phase of NF-κB activation during infection of intact cells may occur due to the combination of cellular activation of IKKα and enhanced direct interactions between either intracellular rickettsiae or a rickettsial factor with inactive cytoplasmic NF-κB.
The specific interactions of Rickettsia organisms with the host EC surface to orchestrate the events resulting in IKK/NF-κB activation remain to be elucidated in further detail. Thus far, the identities of a potential host cell receptor or a major rickettsial component(s) serving to trigger the upstream signaling pathways are not known. Initial examination with inactivated R. rickettsii revealed that the exposure of host cell surface to live bacteria is necessary and a heat-sensitive protein is responsible for the onset of endothelial activation. In R. rickettsii, the major immunodominant surface-exposed proteins are the outer membrane proteins OmpA and OmpB, of which OmpA is critical for initial adhesion to the host cell membrane (26). Further investigation of OmpA involvement in infection-induced cell signaling is particularly important in light of the finding that outer membrane proteins of Bartonella henselae are major pathogenic factors for the production of EC responses (11). Bacterial LPS is known to be a strong inducer of NF-κB in a variety of cell types (2, 12). Although it remains a possibility, a potential role for rickettsial LPS is somewhat unlikely considering its significantly lower endotoxin activity in comparison to classically active LPS from Escherichia coli or Salmonella spp. (13) and the existing evidence for noninvolvement in in vitro endothelial responses characterized thus far (30, 31, 34). Moreover, recent data from our laboratory show that adsorption of LPS by incubation with either polymyxin B-agarose beads (30) or a monoclonal antibody to R. rickettsii LPS has no effect on the activation of IKK. A crude preparation of rickettsial LPS, which was much less effective in inducing the expression and activity of tissue factor in cultured ECs as expected, also had no apparent effect on IKK activity, whereas E. coli (O111:B4) LPS triggered potent induction of both tissue factor and IKK activities (Fig. (Fig.4)4) (S. K. Sahni and E. Rydkina, unpublished observations). Another possibility being explored in our laboratory is that R. rickettsii-induced endothelial signaling, especially late during infection, may be an indirect response due to the production of secondary inducers such as cytokines, e.g., TNF-α or IL-1.
Since NF-κB plays a central role in acute and chronic inflammation, signaling pathways that regulate its activity have become a focal point as molecular targets for the development of new therapeutic compounds (23). Activation of NF-κB is essential for preventing apoptosis and ensuring the survival of ECs during R. rickettsii infection and plays an important role in triggering infection-induced responses of the vascular endothelium by stimulating the expression of chemokines IL-8 and monocyte chemoattractant protein 1 (3; D. R. Clifton, H. Huyck, G. Pryhuber, R. S. Freeman, L. A. Sporn, and S. K. Sahni, Abstr. Am. Soc. Rickettsiol., abstr. 79, p. 58, 2001). Thus, inhibition of IKK and NF-κB by using nonsteroidal anti-inflammatory agents may provide attractive targets for novel antirickettsial therapies. Our data suggest that inhibition of R. rickettsii-induced activation of IKKα and IKKβ with aspirin or parthenolide is sufficient to effectively attenuate the early transient NF-κB response. This may reduce the ensuing inflammatory reactions and risk of vascular dysfunction associated with rickettsial infections. Sesquiterpene lactones such as parthenolide are amenable for use in treating infections and inflammation of the skin and other organs (18). Studies have also shown that aspirin exhibits previously unrecognized antibacterial effects mediated by reduction of hematogenous bacterial dissemination and embolism during Staphylococcus aureus endocarditis (24) and inhibition of chlamydial growth in human ECs (47).
In summary, infection of human ECs with viable R. rickettsii induces IKKα and IKKβ activation, an important signaling event which leads to the phosphorylation and proteolysis of IκBα and translocation of active NF-κB dimers to the nucleus. The steady-state expression levels of key NF-κB proteins p65 and p50 and inhibitor protein IκBβ, however, remain relatively unchanged. The anti-inflammatory compounds aspirin and parthenolide inhibit Rickettsia-induced IKK phosphorylation of IκBα and nuclear translocation of NF-κB, suggesting that at least the early signaling occurs through this established pathway. Such action could effectively disrupt the production of NF-κB-dependent inflammatory mediators. These findings thus provide new insights into the molecular basis of NF-κB regulation during R. rickettsii infection and suggest that newly developed specific inhibitors of IKK activity may not only serve as anti-inflammatory agents but also as efficacious therapies to prevent vascular damage and reduce morbidity in human infections.
We thank Li Hua Rong for cultures of ECs; David J. Silverman for providing R. rickettsii; Edward Schwarz for providing plasmids containing IκBα, IκBβ, and p65 cDNA; Albert Baldwin, Jr., for providing the plasmid containing p105/p50 cDNA; P. J. Simpson-Haidaris for providing the GAPDH probe for Northern analysis; David H. Walker for providing monoclonal antibody to R. rickettsii LPS; and Minh-Doan T. Nguyen and Brian J. Rybarczyk for helpful discussions.
This study was supported in part by grants AI40689 and HL30616 from the National Institutes of Health, Bethesda, Md.
Editor: J. B. Bliska