Campylobacter jejuni infection of differentiated THP-1 macrophages results in interleukin 1{beta} release and caspase-1-independent apoptosis

Amy M. Siegesmund, Michael E. Konkel, John D. Klena and Philip F. Mixter

School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4234, USA

Correspondence
Philip F. Mixter
pmixter{at}wsu.edu


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Apoptosis induction of host macrophages has emerged as a common virulence mechanism among bacterial pathogens. Infection with Campylobacter jejuni is a leading cause of gastroenteritis worldwide and is characterized by an acute inflammatory response in the small intestine. The authors used the human monocytic cell line THP-1 to examine apoptosis induction and pro-inflammatory cytokine production during C. jejuni infection. Flow cytometric analysis revealed that 48 h after inoculation, a C. jejuni wild-type isolate induced apoptosis in 63 % of THP-1 cells while only 34 % of cells inoculated with a ciaB mutant, which does not secrete the Cia (Campylobacter invasion antigens) proteins, underwent apoptosis. Complementation of the ciaB mutant resulted in levels of apoptosis similar to those induced by the C. jejuni wild-type isolate, suggesting that the Cia proteins have a role in apoptosis induction. It was shown that a proteinase K- and heat-stable component of C. jejuni also stimulated THP-1 apoptosis. Inoculation with a C. jejuni gmhD mutant indicated that lipooligosaccharide was not the stimulatory molecule. Immunoblot and ELISA analyses revealed that C. jejuni infection stimulated the synthesis, processing and secretion of interleukin 1{beta} (IL-1{beta}). Inhibition of caspase 1 activity eliminated IL-1{beta} processing and secretion, but did not affect apoptosis induction. In addition, treatment of cells with a caspase-9-specific inhibitor did not affect apoptosis induction, arguing against activation of an apoptotic pathway dependent on either caspase 1 or 9 activation. Collectively, these data suggest that the inoculation of macrophages with C. jejuni results in the processing of IL-1{beta} and apoptosis through different regulatory pathways. Furthermore, these data argue that C. jejuni may use a mechanism distinct from Salmonella typhimurium and Shigella flexneri to initiate macrophage apoptosis and release of IL-1{beta}.


Abbreviations: ADP-HEP, ADP-L-glycero-D-manno-heptose; Cia, Campylobacter invasion antigens; IL-1{beta}, interleukin 1{beta}; LOS, lipooligosaccharide; PI, propidium iodide; PMA, phorbol 12-myristate 13-acetate; WCL, whole-cell lysates


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Apoptosis, or programmed cell death, is necessary for maintaining tissue homeostasis. The pathways leading to apoptosis are many, but fall into two broad categories, caspase-dependent and caspase-independent. Caspases are cysteine proteases that become activated by proteolytic cleavage. There are several pathways that can lead to the activation of caspases, including ligation of death receptors and induction of mitochondrial membrane permeabilization (MMP). MMP results in the release of pro-apoptotic factors such as cytochrome c. Once released, cytochrome c binds to and activates apoptotic protease-activating factor 1 (Apaf-1), forming a complex known as the apoptosome. Pro-caspase 9 is recruited to the apoptosome and is autoactivated (Marsden et al., 2002). Caspase 9 bound to the apoptosome can activate downstream effector caspases, ultimately resulting in DNA fragmentation and cell death. MMP also results in the release of factors that can induce apoptosis independent of caspase activation. Release of factors such as apoptosis-inducing factor (AIF) and endonuclease G can initiate an apoptotic death without caspase activation (Li et al., 2001).

Although apoptosis was initially characterized as a process essential to development and homeostasis in multicellular organisms, data in recent years have demonstrated that during bacterial infection apoptosis induction of host macrophages is a virulence mechanism utilized by pathogens. The induction of host macrophage apoptosis by the enteric pathogens Salmonella enterica serovar Typhimurium (Sal. typhimurium), Shigella flexneri and Yersinia enterocolitica has been the focus of a great deal of research in the last decade, and significant progress has been made in understanding the role of apoptosis induction in the pathogenesis of each of these organisms. Data indicate that during Y. enterocolitica infection, macrophage apoptosis induction is a mechanism for avoiding an inflammatory response (Ruckdeschel et al., 2001). In contrast, it appears that during Sal. typhimurium and Sh. flexneri infection, induction of macrophage apoptosis is essential for inciting host inflammation (Hersh et al., 1999; Zychlinsky et al., 1994). Interestingly, the inhibition or induction of inflammation in these infections appears to serve a common pathogenic goal – the spread of infection from the intestine to deeper tissues. Another commonality among these infections is that apoptosis induction is dependent on type III secretion. Type III secretion systems are used by a variety of Gram-negative pathogens to deliver proteins into host cells. During Y. enterocolitica infection, the type III protein YopP inhibits NF-{kappa}B signalling in macrophages and results in macrophage apoptosis and suppression of the inflammatory response (Ruckdeschel et al., 2001). In contrast, during Sal. typhimurium and Sh. flexneri infection the type III proteins SipB and IpaB directly activate caspase 1, resulting in macrophage apoptosis and the release of pro-inflammatory cytokines such as interleukin 1{beta} (IL-1{beta}) (Chen et al., 1996; Monack et al., 1996). Collectively, these data indicate that pathogen-induced macrophage apoptosis is a mechanism of exploiting host signalling pathways to establish an infection.

Infection with Campylobacter jejuni is one of the most common bacterial causes of gastroenteritis worldwide, reported more frequently than infection by either Sal. typhimurium or Escherichia coli O157 : H7 (Mead et al., 1999). In contrast to other enteric infections, C. jejuni-mediated enteritis is often a self-limiting disease characterized by fever, abdominal cramping and acute intestinal inflammation leading to diarrhoea containing blood and leukocytes. Although the clinical pathology associated with C. jejuni infection has been characterized, the bacteria–host interactions involved in inciting the host inflammatory response are less well understood. Based on data indicating that macrophage apoptosis induced by bacterial pathogens is involved in generating inflammation and the fact that C. jejuni infection is characterized by an acute inflammatory response, we wanted to determine if the inflammation associated with C. jejuni infection was associated with the induction of host macrophage apoptosis. In addition, we examined C. jejuni products for their contribution to apoptosis induction, in particular the secreted Campylobacter invasion antigens (Cia proteins) and lipooligosaccharide (LOS).

In vitro experiments have revealed that C. jejuni synthesizes unique proteins when cultured in the presence of eukaryotic cells, a subset of which is secreted (Konkel et al., 1999; Rivera-Amill et al., 2001). These secreted Cia proteins are required for the maximal invasion of host epithelial cells by C. jejuni (Konkel et al., 1999; Rivera-Amill et al., 2001). A mutation in the gene that encodes a 73 kDa secreted protein, CiaB, eliminates secretion of the other Cia proteins and results in a non-invasive phenotype (Konkel et al., 1999). CiaB is detected in the cytoplasm of intestinal epithelial cells, and given that it lacks a cleavable signal sequence and is exported across the inner and outer membranes without a periplasmic intermediate (Rivera-Amill et al., 2001), it conforms to the criteria of proteins exported via a type III system. Perhaps more importantly, inoculation of colostrum-deprived piglets with a C. jejuni ciaB mutant resulted in delayed onset of diarrhoea compared to the inoculation of piglets with the C. jejuni wild-type isolate (Konkel et al., 2001). Based on the fact that other enteric pathogens secrete proteins that have key roles in host cell apoptosis induction, we investigated whether the Cia proteins play a role in apoptosis induction during C. jejuni infection.

Lipopolysaccharides (LPS) and LOS are the major components of the outer membrane in Gram-negative bacteria. LPS has three distinct structural components: lipid A, which serves as the membrane anchor; a core composed of heterogeneous glycoses; and the somatic O antigen (O Ag) composed of a repeating unit of one or more glycosyl residues attached covalently to the core. Lipid A–core moieties lacking O Ag are referred to as LOS. Early reports indicated that the LPS of C. jejuni is similar to that of Haemophilus and Neisseria spp., characterized as being of low molecular mass and lacking detectable amounts of O Ag (Logan & Trust, 1984). High-molecular-mass carbohydrate material present in some isolates of C. jejuni has recently been shown to represent capsular polysaccharide and not O Ag (Karlyshev et al., 2000). Thus, we will refer to this molecule throughout the remainder of the text as LOS. A novel seven-carbon sugar, ADP-L-glycero-D-manno-heptose (ADP-HEP), is a conserved component in the LOS/LPS core structures of most Gram-negative bacteria. An epimerase encoded by the gene gmhD is responsible for conversion of the precursor ADP-D-glycero-D-manno-heptose to the final incorporated form (Valvano et al., 2000). Mutations in gmhD result in a truncated core oligosaccharide, easily detectable by SDS-PAGE (Sirisena et al., 1994). Characterization of these mutants reveals that they are extremely sensitive to bile salts, hydrophobic antibiotics and dyes (Nikaido & Vaara, 1985). In order to assess the role of full-length LOS in apoptosis induction, a C. jejuni gmhD mutant was created and compared to the wild-type C. jejuni strain.

We used differentiated THP-1 cells to study the interaction of C. jejuni with human macrophages. We present data indicating that during infection C. jejuni induces apoptosis of differentiated THP-1 cells. We also demonstrate that production of the Cia proteins as well as a proteinase K- and heat-resistant bacterial component are required for maximal apoptosis induction. Our data indicate that caspase 1 is activated during C. jejuni infection, leading to secretion of the pro-inflammatory cytokine IL-1{beta}. However, neither caspase 1 nor caspase 9 is required for C. jejuni-stimulated apoptosis. These data indicate that the mechanism used by C. jejuni to induce IL-1{beta} release and apoptosis is different from that of Sal. typhimurium and Sh. flexneri. The significance of macrophage apoptosis to C. jejuni-mediated enteritis is also discussed.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial isolates and growth conditions.
The C. jejuni F38011 wild-type isolate was cultured on Mueller–Hinton agar plates containing 5 % (v/v) bovine citrated blood (MHB) under microaerobic conditions at 37 °C. C. jejuni ciaB and gmhD mutants were cultured on MHB plates supplemented with 200 µg kanamycin ml-1. The C. jejuni ciaB mutant transformed with pMEK100 was cultured on MHB plates supplemented with 12·5 µg tetracycline ml-1. All Campylobacter isolates were subcultured every 24–48 h.

Isolation and characterization of the C. jejuni gmhD isogenic mutant.
The gmhD gene in C. jejuni F38011 was disrupted by homologous recombination via a single crossover event between the gmhD gene on the chromosome and an internal fragment of the cloned gene on a suicide vector using methods described previously (Konkel et al., 1999). The correct location of the crossover event on the chromosome was confirmed by PCR. The LOS phenotype was confirmed by GC-MS (data not shown).

Preparation of bacterial whole-cell lysates (WCL).
The C. jejuni F38011 wild-type isolate and isogenic ciaB mutant were grown overnight on MHB plates or MHB plates supplemented with kanamycin. Bacteria were harvested and pelleted by centrifugation at 6000 g. The bacterial pellets were washed twice and resuspended in phosphate buffered saline (PBS). Bacteria were lysed by sonication (5x30 s bursts with 30 s intervals between each burst). Sonicates were centrifuged at 3700 g for 10 min at 4 °C. The supernatants were removed and further purified by centrifugation at 10 000 g for 10 min at 4 °C. Clarified cell lysates were then subjected to proteinase K digestion and heat treatment. For proteinase K digestion, 100 µg proteinase K ml-1 (Sigma) was added to the lysate and incubated for 45 min at 65 °C. Following proteinase K digestion, lysates were boiled for 10 min and any precipitants were removed by centrifugation. Electrophoretic gel analyses of proteinase K-treated samples indicated effective protein digestion (not shown). Protein concentration of lysate preparations was determined using the BCA Assay (Pierce Endogen). Purified Sal. typhimurium LPS was purchased from Sigma.

Tissue culture and differentiation of THP-1 cells.
Stock cultures of THP-1 cells (human monocyte, ATCC TIB-202) were grown in RPMI 1640 (Mediatech) supplemented with 10 % (v/v) fetal bovine serum, 4·5 g glucose l-1, 10 mM HEPES, 1·5 g sodium pyruvate l-1 and 0·05 mM 2-mercaptoethanol. Cultures were maintained at a density of approximately 2x105 cells ml-1 at 37 °C in a humidified 7 % (v/v) CO2 incubator and were passaged every 3–4 d. Cells were resuspended in THP-1 culture medium supplemented with phorbol 12-myristate 13-acetate (PMA; Sigma) at a final concentration of 10 ng PMA ml-1 to induce differentiation (Tsuchiya et al., 1982). Cells were dispensed into tissue culture plates at a density of approximately 1x106 cells per well and incubated for 24 h in a 37 °C humidified 7 % (v/v) CO2 incubator. After incubation, PMA-containing medium was aspirated and adherent (differentiated) cells were washed once with THP-1 culture medium. Differentiated cells were then incubated in THP-1 culture medium for an additional 24 h prior to inoculation.

Inoculation protocol.
C. jejuni were harvested from MHB plates in PBS and pelleted by centrifugation at 6000 g at 4 °C. The bacterial pellets were washed once in PBS and resuspended in RPMI 1640 containing 1 % (v/v) bovine calf serum (BCS). Bacterial suspensions were diluted in RPMI 1640 containing 1 % (v/v) BCS and optical density (OD540) was determined. Cell monolayers were washed three times with RPMI 1640 and bacterial suspensions added in a volume sufficient to achieve an m.o.i. of 200 c.f.u. per cell. Plates were centrifuged at 400 g to synchronize bacteria–host cell interactions. In some experiments, bacterial suspensions were incubated in RPMI 1640 containing 1 % (v/v) BCS supplemented with either 250 µg gentamicin ml-1 (Sigma) or 128 µg chloramphenicol ml-1 (Sigma) for 3 h prior to use in infection. In experiments utilizing whole-cell lysates (WCL), a volume of lysate equivalent to 25 µg protein (diluted in RPMI 1640 containing 1 %, v/v, BCS) was added to cell monolayers. In experiments using caspase inhibitors, cells were incubated with the caspase-1-specific inhibitor Z-YVAD-FMK (R&D Systems) or the caspase-9-specific inhibitor Z-LEHD-FMK (R&D Systems) for 3 h prior to inoculation.

Flow cytometric analysis of apoptosis induction.
At each time point after inoculation, the medium was aspirated and RPMI 1640 containing 1x trypsin-EDTA (Invitrogen) was added to each well. After a 10 min incubation at 37 °C, cells were removed from wells by gentle scraping with a cell scraper. Cells were dispensed into cold tubes with PBS containing 1 % (w/v) bovine serum albumin (BSA) and 0·01 % (w/v) sodium azide (Sigma). After cells were washed with PBS containing 0·01 % (w/v) sodium azide, pellets were resuspended in annexin binding buffer (10 mM HEPES buffer, 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2·5 mM CaCl2) containing annexin-V-FITC (Caltag Laboratories). Following a 10 min incubation, propidium iodide (PI) was added to a final concentration of 5 µg ml-1. Samples were incubated on ice for 30–60 min prior to flow cytometry analysis using a FACSCalibur flow cytometer (BD Biosciences). For each sample, 10 000 events were collected and analysed using CellQuest software (BD Biosciences). Uninoculated THP-1 cells stained with annexin-V-FITC and PI were used to determine background levels of apoptosis. Apoptotic events were defined as those staining positively (above background levels) for annexin-V-FITC and negatively for PI. Data are presented as the percentage of total ungated events staining positively for annexin-V-FITC and negatively for PI.

Immunoblot analysis of IL-1{beta}.
At the designated times post-inoculation, cells were harvested from tissue culture plates and lysed in lysis buffer [25 mM Tris, 137 mM NaCl, 10 % (v/v) glycerol, 0·5 % (w/v) sodium deoxycholate, 1 % (v/v) Igepal CA-630, 2 mM EDTA] containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 30 µg aprotonin ml-1, 1 mM sodium orthovanadate). Lysates were clarified by sonication (5x45 s bursts with 15 s intervals between each burst). Insoluble material was removed by centrifugation as described above. Lysates were separated on SDS-12 % polyacrylamide gels and transferred to nitrocellulose (Bio-Rad) in buffer containing 25 mM Tris, 192 mM glycine and 20 % (v/v) methanol. Membranes were incubated with anti-human IL-1{beta} antibodies (Pierce-Endogen) followed by goat anti-mouse horseradish-peroxidase-conjugated secondary antibody (Sigma). Reactive proteins were visualized by chemiluminescence (SuperSignal, Pierce-Endogen). Following exposure to X-ray film, an image was captured using a Fluor-S MultiImager (Bio-Rad). Signal intensity was quantified as intensity units per mm2 (volume units) using Quantity One software (version 4, Bio-Rad). Fold induction was calculated as the increase in sample volume units compared to volume units of the uninfected control (background).

IL-1{beta} ELISA.
The levels of IL-1{beta} produced during C. jejuni interaction with host cells were quantified using the Matched Antibody Pair ELISA specific for IL-1{beta} (Pierce Endogen). Assays were performed according to the manufacturer's specifications. Values for samples were determined using a standard curve generated on the day of each assay. The lower limit of ELISA IL-1{beta} detection was 78 pg IL-1{beta} ml-1. Values represent mean±standard deviation of three independent experiments.

Statistics.
Data are presented as the mean±standard deviation of n’ experiments. The statistical difference was determined using the paired Student's t test. A P-value <0·05 was considered statistically significant.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
C. jejuni induces apoptosis of differentiated human monocytes
Apoptosis induction by bacterial pathogens has emerged as a common theme in bacterial pathogenesis (Chen et al., 1996; Monack et al., 1996, 1997). Due to the ability of C. jejuni to translocate across the intestinal epithelium (Konkel et al., 1992) we were interested in examining the interaction of C. jejuni with cells found in the lamina propria, specifically macrophages. Differentiation of the human monocytic cell line THP-1 was induced with PMA, resulting in a macrophage morphology (Tsuchiya et al., 1982). The differentiated cells were inoculated with the C. jejuni F38011 wild-type isolate, the isogenic ciaB mutant, the F38011 wild-type isolate killed with gentamicin or the isogenic ciaB mutant killed with gentamicin (Fig. 1). At the indicated time points, levels of apoptosis induction were assessed by flow cytometry. At 3 and 6 h post-inoculation, the levels of apoptosis induced by the F38011 wild-type isolate, ciaB mutant and gentamicin-treated bacteria were similar to the background level (sham). Beginning at 24 h post-inoculation, the level of apoptosis induced by the C. jejuni wild-type isolate increased over background and over the level of apoptosis induced by the ciaB mutant. This trend continued over the duration of the time-course, with the level of apoptosis induced by C. jejuni wild-type isolate reaching 63 % at 48 h post-inoculation. This level of apoptosis was significantly higher (P<0·05) than that induced by the ciaB mutant, which was 34·2 % at 48 h post-inoculation. It is important to note that although the level of apoptosis induced by the ciaB mutant was significantly lower than wild-type, the level was significantly higher than that induced by gentamicin-treated bacteria and background. Taken together, these data indicated that maximal apoptosis of THP-1 cells during C. jejuni inoculation was dependent on secretion of the Cia proteins and other C. jejuni-specific components.



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Fig. 1. C. jejuni induces apoptosis of differentiated human monocytes. Differentiated THP-1 cell monolayers were inoculated with either the C. jejuni F38011 wild-type isolate ({circ}), an isogenic ciaB mutant (CiaB-, {bullet}), or C. jejuni treated with 250 µg gentamicin ml-1 prior to inoculation ({square}, F38011+gentamicin; {blacksquare}, CiaB-+gentamicin). Uninoculated cells (sham, {triangleup}) served as a negative control. At the indicated times post-inoculation, cells were harvested, stained with annexin-V-FITC and PI, and analysed by flow cytometry. The proportion of ungated events staining positively for annexin-V-FITC and negatively for PI at each time point is shown (mean±SD of three independent experiments).

 
To confirm the role of the Cia proteins in apoptosis induction, THP-1 monolayers were inoculated with either the C. jejuni F38011 wild-type isolate, the ciaB mutant, or the ciaB mutant transformed with pMEK100. The pMEK100 recombinant plasmid contains a 2248 bp fragment harbouring the entire C. jejuni F38011 ciaB gene under control of its native promoter (Rivera-Amill et al., 2001). Forty-eight hours post-inoculation, the levels of apoptosis were quantified by flow cytometric analysis (Fig. 2). Consistent with earlier results, the level of apoptosis induced by the ciaB mutant was significantly higher than the background level, but was significantly lower than the level of apoptosis induced by C. jejuni wild-type isolate. Complementation of the ciaB mutant in trans resulted in a level of apoptosis comparable to that induced by C. jejuni wild-type isolate, confirming our previous observation that secretion of the Cia proteins was required for maximal apoptosis induction of THP-1 cells.



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Fig. 2. Secretion of the Cia proteins is required for maximal apoptosis induction. Differentiated THP-1 cell monolayers were inoculated with either the C. jejuni F38011 wild-type isolate (F38011), an isogenic ciaB mutant (CiaB-), or a complemented (pMEK100) ciaB mutant. Uninoculated cells (sham) served as the negative control. Forty-eight hours post-inoculation, cells were harvested, stained with annexin-V-FITC and PI, and analysed by flow cytometry. The proportion of ungated data staining positively for annexin-V-FITC and negatively for PI is shown (mean±SD of three independent experiments).

 
Contribution of bacterial components to C. jejuni-mediated apoptosis
Although necessary for maximal apoptosis induction in vitro, secretion of the Cia proteins alone did not appear to be sufficient to induce a level of apoptosis comparable to wild-type. The levels of apoptosis induced by the ciaB mutant at 24, 36 and 48 h post-inoculation were significantly lower than those induced by the wild-type isolate, but were significantly higher than background (Figs 1 and 2). In addition, the level of apoptosis induced by gentamicin-killed wild-type C. jejuni was significantly higher than background 48 h post-inoculation. These data suggested that, in addition to the Cia proteins, other bacterial components contributed to apoptosis induction during C. jejuni inoculation.

A previous study indicated that outer-membrane proteins stimulated apoptosis of chicken lymphocytes during infection with C. jejuni (Zhu et al., 1999). To investigate this possibility as well as determine if viable C. jejuni were required for apoptosis induction, we co-cultured THP-1 monolayers with bacterial WCL preparations generated from the C. jejuni wild-type isolate or ciaB mutant. To determine the nature of the molecule able to induce apoptosis of THP-1 cells, we also used bacterial WCL preparations that had been treated with proteinase K and heat prior to co-culture with THP-1 cells. Co-culture of THP-1 monolayers with WCL preparations from both the F38011 wild-type and the ciaB mutant resulted in levels of apoptosis induction similar to the level induced by the viable ciaB mutant, but below the level induced by the C. jejuni wild-type isolate (Fig. 3b). Furthermore, treatment of the WCL preparations with proteinase K and heat did not significantly reduce the amount of apoptosis, indicating that a component of C. jejuni resistant to proteinase K and heat was able to stimulate apoptosis of THP-1 cells.



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Fig. 3. Contribution of bacterial components to C. jejuni-induced apoptosis. (a) Differentiated THP-1 cell monolayers were infected with either the C. jejuni F38011 wild-type isolate or the gmhD (GmhD-) mutant. Uninoculated cells (sham) served as the negative control. Twenty-four hours post-inoculation, cells were harvested, stained with annexin-V-FITC and PI, and analysed by flow cytometry. The proportion of ungated data staining positively for annexin-V-FITC and negatively for PI is shown (mean±SD of three independent experiments). (b) Differentiated THP-1 monolayers were inoculated with either the C. jejuni F38011 wild-type isolate or the ciaB mutant (CiaB-). Monolayers were inoculated with either viable bacteria (grey bars), bacterial WCL (black bars), or bacterial WCL treated with proteinase K and heat (white bars). Twenty-four hours post-inoculation, levels of apoptosis were quantified as described above. The mean±SD of three independent experiments is shown.

 
LOS is a proteinase K- and heat-resistant molecule that could play a role in apoptosis induction. To determine the possible role of LOS in C. jejuni-mediated apoptosis, differentiated THP-1 monolayers were inoculated with either the C. jejuni wild-type isolate or a C. jejuni gmhD mutant, the latter of which contains a severely truncated LOS due to the failure of this isolate to produce and incorporate ADP-HEP (not shown). Twenty-four hours post-inoculation, the level of apoptosis induced by the gmhD mutant was nearly identical to that induced by the C. jejuni wild-type isolate, indicating that native LOS does not contribute to apoptosis induction of THP-1 cells (Fig. 3a). These results, in conjunction with our earlier findings, indicate that C. jejuni apoptosis is stimulated by secretion of the Cia proteins in addition to a proteinase K- and heat-stable bacterial component distinct from native LOS.

Inoculation with C. jejuni results in synthesis and secretion of IL-1{beta}
Caspase 1 has a role not only in apoptosis induction, but also in the processing of pro-inflammatory cytokines such as IL-1{beta}. Pro-IL-1{beta} is synthesized as a precursor of approximately 31 kDa (Andrei et al., 1999). Pro-IL-1{beta} is cleaved to its mature, secreted form by caspase 1. To investigate whether C. jejuni-induced THP-1 cell apoptosis was associated with synthesis of pro-IL-1{beta}, caspase 1 activation, and release of IL-1{beta}, differentiated THP-1 monolayers were inoculated with either the C. jejuni wild-type isolate or the ciaB mutant. Twenty-four hours post-inoculation, THP-1 cells were harvested and lysates generated as described in Methods. Immunoblot analysis of inoculated THP-1 cell lysates revealed a band of the predicted size for pro-IL-1{beta} (Fig. 4). A 17 kDa band corresponding to mature IL-1{beta} was not detected in any of the cell lysates. Incubation of THP-1 monolayers with either the C. jejuni wild-type isolate, the ciaB mutant, or 1 µg of Sal. typhimurium LPS ml-1 resulted in similar levels of IL-1{beta} production. Compared to the uninoculated control, the levels of pro-IL-1{beta} induced by the C. jejuni wild-type isolate, the ciaB mutant, and 1 µg of Sal. typhimurium LPS ml-1 were increased approximately 11-fold (data not shown).



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Fig. 4. Synthesis of pro-IL-1{beta} during C. jejuni infection. Differentiated THP-1 monolayers were inoculated with either the C. jejuni F38011 wild-type isolate or the ciaB mutant. Twenty-four hours post-inoculation, THP-1 cells were harvested and lysates prepared as described in Methods. Lysates were resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblot analysis was performed using IL-1{beta}-specific antibodies. Lanes: 1, uninoculated THP-1; 2, THP-1 treated with 1 µg ml-1 of purified Sal. typhimurium LPS; 3, THP-1 inoculated with the C. jejuni wild-type isolate; 4, THP-1 inoculated with ciaB mutant.

 
The amount of mature, secreted IL-1{beta} from infected THP-1 cells was also quantified. Twenty-four hours post- inoculation, supernatants were collected from THP-1 monolayers inoculated with either the C. jejuni wild-type isolate, the ciaB mutant or Sal. typhimurium LPS. ELISA analyses of secreted IL-1{beta} indicated that mature IL-1{beta} was released from THP-1 cells inoculated with either F38011 (2029±220 pg IL-1{beta} ml-1), the ciaB mutant (1963±146 pg IL-1{beta} ml-1) or Sal. typhimurium LPS (1716±127 pg IL-1{beta} ml-1), but not from the uninoculated control.

These data indicate that inoculation of THP-1 cells with either the C. jejuni wild-type isolate, the ciaB mutant or Sal. typhimurium LPS results in synthesis of pro-IL-1{beta}, activation of caspase 1, and secretion of IL-1{beta}.

Inhibition of caspase 1 or caspase 9 does not significantly affect C. jejuni-induced apoptosis
Based on our data indicating that caspase 1 was activated during C. jejuni inoculation, we investigated whether apoptosis induction was dependent on caspase 1 activity. Differentiated THP-1 monolayers were treated with the caspase 1-specific inhibitor Z-YVAD-FMK for 3 h prior to inoculation with wild-type C. jejuni F38011. Twenty-four hours post-inoculation the levels of apoptosis induction were assessed by flow cytometry. To verify inhibition of caspase 1, supernatants were collected and secreted IL-1{beta} was quantified. Inhibition of caspase 1 by Z-YVAD-FMK eliminated secretion of IL-1{beta} detected by ELISA (data not shown), but did not significantly affect apoptosis induction by wild-type C. jejuni (Fig. 5). We also examined the effect of caspase 9 inhibition to determine if C. jejuni induced apoptosis of THP-1 cells in a mitochondria- and caspase-dependent manner. THP-1 monolayers were treated with the caspase 9 inhibitor Z-LEHD-FMK and inoculated as described above. Inhibition of caspase 9 activity was confirmed by a colorimetric caspase 9 activity assay (data not shown). Inhibition of caspase 9 did not significantly block apoptosis induction, arguing against a pathway of apoptosis induction dependent on both the mitochondria and caspases. Collectively, these data indicate that the pathway utilized by C. jejuni to induce apoptosis is different from that of Sal. typhimurium and Sh. flexneri, stimulating caspase 1 activity and release of IL-1{beta} independently of apoptosis induction.



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Fig. 5. Effect of caspase inhibition on C. jejuni-induced apoptosis of THP-1 cells. Differentiated THP-1 monolayers were treated with PBS (sham) or inoculated with the C. jejuni F38011 wild-type isolate in either the absence (untreated) or presence of caspase inhibitors specific for either caspase 1 (Z-YVAD-FMK) or caspase 9 (Z-LEHD-FMK). Monolayers were treated with caspase inhibitors at a final concentration of 20 µM for 3 h prior to inoculation. Twenty-four hours post-inoculation, cells were harvested, stained with annexin-V-FITC and PI, and analysed by flow cytometry. The proportion of ungated data staining positively for annexin-V-FITC and negatively for PI is shown (mean±SD of three independent experiments).

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Although acute intestinal inflammation is one of the hallmarks of C. jejuni-mediated enteritis, it remains unclear how the interaction of C. jejuni with host cells elicits such a response. The inflammatory response associated with infection by the enteric pathogens Sal. typhimurium and Sh. flexneri has been well characterized. In both infections, apoptosis induction of host macrophages is key to inciting the inflammatory response and is necessary for disease progression. The present study utilized an in vitro system to examine the interaction of C. jejuni with human macrophages. We determined that inoculation with C. jejuni caused the release of the pro-inflammatory cytokine IL-1{beta} in 3 h and apoptosis of human THP-1 macrophages during the 48 h after inoculation. We demonstrated that maximal apoptosis induction depends on a bacterial component that is resistant to proteinase K and heat as well as secretion of the Cia proteins. Furthermore, we showed that although caspase 1 is activated and leads to the production of IL-1{beta}, the activation of caspase 1 did not contribute to C. jejuni-induced apoptosis. These findings suggest that C. jejuni induces apoptosis and IL-1{beta} release in a manner distinct from enteric pathogens such as Sal. typhimurium and Sh. flexneri.

We quantified the ability of C. jejuni to induce host cell apoptosis by flow cytometric analysis of C. jejuni-inoculated THP-1 cells. When THP-1 monolayers were inoculated with C. jejuni, we found that the C. jejuni wild-type isolate was able to induce apoptosis of 63 % of THP-1 cells over a 48 h time-course. In contrast, only 34 % of THP-1 cells inoculated with a C. jejuni ciaB mutant underwent apoptosis during the same time-course, indicating a role for the Cia proteins in apoptosis induction (Fig. 1). We confirmed a role for the secreted Cia proteins in apoptosis induction by inoculating THP-1 cells with a complemented ciaB mutant strain. Twenty-four hours post-inoculation, the level of apoptosis induced by the complemented strain was comparable to the level induced by the wild-type isolate, confirming the role of the Cia proteins in apoptosis induction (Fig. 2). The requirement of Cia proteins for maximal apoptosis induction was not unexpected, as other Gram-negative pathogens secrete proteins involved in apoptosis induction. More specifically, SipB from Sal. typhimurium and IpaB from Sh. flexneri are required for apoptosis induction of host macrophages (Chen et al., 1996; Monack et al., 1996). In contrast to IpaB and SipB, elimination of Cia protein secretion by eliminating a functional ciaB gene reduced, but did not eliminate, apoptosis induction of host macrophages. Another striking difference between Cia proteins, IpaB and SipB is that mutation of ciaB eliminates secretion of all the Cia proteins. As the ciaB mutant is deficient in secretion of all the Cia proteins (Konkel et al., 1999), it is possible that the reduction in apoptosis induction observed with the ciaB mutant is not due to a lack of CiaB directly, but to a lack of one of the other Cia proteins. Functional studies of the Cia proteins are an ongoing area of investigation.

As mentioned above, when THP-1 cells were infected with the ciaB mutant, we did not observe a complete elimination of apoptosis induction. We found that co-culture of WCL preparations generated from either the C. jejuni wild-type isolate or the ciaB mutant induced levels of apoptosis similar to those induced by the viable ciaB mutant. This finding was in agreement with our earlier experiments demonstrating that bacterial components and secretion of the Cia proteins by viable C. jejuni were required for maximal apoptosis induction (Figs 1 and 2). Treatment of the WCL with proteinase K and heat did not eliminate their ability to elicit an apoptotic response (Fig. 3). The results of the WCL experiments supported two conclusions. First, although required for maximal apoptosis induction, viable C. jejuni were not essential for apoptosis induction. Second, a bacterial molecule resistant to proteinase K and heat was able to stimulate significant levels of apoptosis of THP-1 cells. This finding argues that a heat-labile bacterial toxin, such as cytolethal distending toxin (Johnson & Lior, 1988), does not contribute significantly to C. jejuni-induced apoptosis of THP-1 cells. Our results differ from those of Zhu et al. (1999) demonstrating that outer-membrane proteins from C. jejuni stimulated apoptosis in chicken lymphocytes and that treatment of the outer-membrane protein preparations with proteinase K eliminated the apoptotic effect. We found that proteinase K and heat treatment did not affect levels of apoptosis induced by WCL preparations. The differences in our results may be due to the differences in bacterial preparations used as stimuli, but are more likely due to the differences in species and cell type. Macrophages, as key components of the innate immune response, express a wide variety of receptors to recognize diverse molecules and are responsive to several different types of bacterial components. Lymphocytes are more restricted in their ability to respond to diverse bacterial molecules. Therefore, it is not unreasonable to expect that the bacterial molecules that stimulate macrophage and lymphocyte apoptosis differ.

Based on our result indicating that apoptosis was stimulated by a bacterial component that was resistant to both proteinase K and heat, we determined the contribution of native LOS to apoptosis induction. Inoculation of THP-1 cells with a deep-rough mutant (gmhD) resulted in levels of apoptosis induction comparable to the wild-type isolate. The gmhD mutation results in a severely truncated LOS molecule, containing only lipid A and the eight-carbon sugar 2-keto-3-deoxyoctolusonic acid (KDO). In contrast, native LOS of C. jejuni is composed of at least two molecules of ADP-HEP and an assortment of glycoses, linked to lipid A-KDO via ADP-HEP. The phenotype of a gmhD mutation is expected to be more severe than a mutation in heptosyl transferase II (encoded by the waaF gene), as strains carrying a waaF mutation would still attach a single ADP-HEP. As anticipated, a recent report has demonstrated the severe pleiotropic nature of a waaF mutant, characterized by a truncated LOS core and loss of ganglioside cross-reactivity associated with the terminal portion of the LOS molecule (Oldfield et al., 2002). Interestingly, the waaF mutation did not affect the high-molecular-mass carbohydrate material thought to be capsule. The data presented here argue against a role for C. jejuni LOS in apoptosis induction of THP-1 cells but indicate that a bacterial component resistant to proteinase K and heat, such as the capsule, is able to stimulate apoptosis of THP-1 cells. Previously, Bacon et al. (2001) showed that a C. jejuni 81-176 mutant lacking the high-molecular-mass glycan in the capsule did not adhere or invade INT 407 epithelial cells optimally and caused diarrhoea in ferrets at reduced rates.

We investigated how C. jejuni-induced macrophage apoptosis might contribute to the intestinal inflammation associated with infection. During Sh. flexneri infection, secretion of SipB results in activation of caspase 1 and subsequent macrophage apoptosis and release of the pro-inflammatory cytokine IL-1{beta}. When THP-1 cells were inoculated with either the C. jejuni wild-type isolate or the ciaB mutant, synthesis of pro-IL-1{beta} was induced (Fig. 4). A recent publication also reported the release of IL-1{beta} by THP-1 cells in response to C. jejuni infection (Jones et al., 2003). Furthermore, we determined that caspase 1 was activated and IL-1{beta} secreted from THP-1 inoculated with either the C. jejuni wild-type isolate or the ciaB mutant (see Results). Although the activation of caspase 1 and the release of IL-1{beta} are similar to what occurs during Sh. flexneri infection, there are significant differences that merit discussion. First, inoculation with both the C. jejuni wild-type isolate and the ciaB mutant resulted in the synthesis and secretion of IL-1{beta}. Strains of Sh. flexneri deficient in production of the type III protein IpaB do not induce secretion of IL-1{beta} (Zychlinsky et al., 1994). Second, infection with Sh. flexneri does not stimulate the synthesis of IL-1{beta}, only its release (Zychlinsky et al., 1994). Additionally, differences in the assembly and secretion of capase-1-activating inflammasome complexes (Tschopp et al., 2003) in the intestinal macrophages induced by either Cia or Ipa proteins could alter kinetics of cytokine release or cell death, leading to differences in disease resolution. As caspase 1 was activated by C. jejuni, we evaluated the contribution of caspase 1 to C. jejuni-mediated apoptosis. Inhibition of caspase 1 eliminated secretion of IL-1{beta} but did not significantly inhibit apoptosis induction (Fig. 5). These results indicated that activation of caspase 1 is required for production of IL-1{beta}, but occurs independently of C. jejuni-induced apoptosis. This finding indicates that the mechanism(s) employed by C. jejuni to induce release of pro-inflammatory cytokines and apoptosis differs from the mechanism used by Sh. flexneri.

As C. jejuni-induced apoptosis was not caspase 1 dependent, we determined if the pathway stimulated by C. jejuni involved the mitochondria and caspase 9. We determined that inhibition of caspase 9 did not affect the levels of apoptosis stimulated by the C. jejuni wild-type isolate (Fig. 5). In addition, we did not detect any caspase 9 activity in C. jejuni-infected THP-1 cells (data not shown). These findings indicated that C. jejuni does not stimulate apoptosis via a pathway involving the apoptosome. However, it is possible that C. jejuni infection stimulates a pathway of apoptosis induction that is mitochondria-dependent but caspase-independent. The anti-apoptotic protein Bcl-2 protects cells from apoptosis by maintaining mitochondrial membrane integrity and preventing the release of pro-apoptotic factors such as cytochrome c. We have found that overexpression of Bcl-2 in Jurkat cells is protective against C. jejuni-mediated apoptosis (data not shown). Based on our data indicating that caspase 9 is not activated during C. jejuni-induced apoptosis we believe that it is unlikely that the protection conferred by Bcl-2 overexpression is due to inhibition of cytochrome c release. Recent data also indicate that Bcl-2 can regulate mitochondria-dependent cell death pathways independently of the apoptosome (Marsden et al., 2002). Therefore, it is possible that C. jejuni stimulates an apoptotic pathway that is dependent on the mitochondria and release of other factors such as apoptosis-inducing factor. Further investigation is required to precisely delineate the apoptotic pathway stimulated during C. jejuni infection.

We have shown that, like other enteric pathogens, C. jejuni induces apoptosis of host macrophages concurrent with the release of pro-inflammatory cytokines. In contrast, we have demonstrated that the pathway stimulated by C. jejuni to induce simultaneous apoptosis induction and IL-1{beta} release appears to be distinct from that of other enteric pathogens. At present it is unclear what purpose macrophage apoptosis might serve in C. jejuni-mediated enteritis. In other systems, the activation of caspase 1 is required for both apoptosis induction and the release of IL-1{beta}. In these scenarios, macrophage apoptosis is thought to facilitate generation of the inflammatory response. As these events can be uncoupled during C. jejuni infection, it is unlikely that macrophage apoptosis is a prerequisite for inflammation. The kinetics of macrophage apoptosis and release of IL-1{beta} also differ in C. jejuni infection compared to other enteric pathogens such as Sh. flexneri. We did not detect significant levels of apoptosis induction until 24 h post-inoculation, with significant IL-1{beta} production detected at 6 h post-inoculation (data not shown). This difference in the timing of apoptosis induction and IL-1{beta} release supports our argument that these events are not dependent on each other. The fact that macrophage apoptosis is not required for generation of the inflammatory response suggests that apoptosis may be a defence mechanism utilized by the host rather than a pathogenic mechanism of C. jejuni. Navarre & Zychlinsky (2000) have proposed that host macrophages undergo apoptosis in response to physiological changes that occur during bacterial infection. By initiating suicide, macrophages would eliminate a potential niche for C. jejuni and would expose the pathogen to immune cells and mediators that could mediate elimination of the infection. Data from Day et al. (2000) suggest that production of catalase by C. jejuni enhances survival within murine and porcine macrophages. Given that most C. jejuni infections are self-limiting, it stands to reason that other immune cells and/or mediators contribute to clearance of the organism.

In summary, we have shown that C. jejuni-stimulated human macrophages are stimulated to undergo apoptosis and release the pro-inflammatory cytokine IL-1{beta}. IL-1{beta} release is dependent on caspase 1. However activation of caspase 1 does not lead to macrophage apoptosis. In addition, C. jejuni does not stimulate an apoptotic pathway dependent on caspase 9. We hypothesize that during C. jejuni infection, permeabilization of the mitochondrial membrane resulting in a mitochondria-dependent pathway of apoptosis may be a defence mechanism of the host aimed at limiting C. jejuni infection. Future studies examining the interaction of C. jejuni with other immune cells and mediators as well as continued examination of the C. jejuni–macrophage interaction will dissect these complex interactions that contribute to C. jejuni-mediated enteritis.


   ACKNOWLEDGEMENTS
 
This work was supported by a grant from the Washington State University (WSU) College of Sciences to A. M. S., by funds from the WSU School of Molecular Biosciences to P. F. M., and by a grant from the NIH (DK 58911) to M. E. K. We appreciate critical discussions with Dr Brian Raphael (WSU School of Molecular Biosciences).


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 7 May 2003; revised 13 October 2003; accepted 25 November 2003.



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