Introduction
Clostridium difficile is the most common cause of antibioticassociated diarrhea and pseudomembranous colitis in humans [12,16-19]. C. difficile produces intestinal damage and diarrhea by releasing two exotoxins: toxin A and toxin B. These toxins have glucosyltransferase activity, which monoglucosylates the small GTPases Rho, Rac, and Cdc42 at threonine 37, triggering actin disaggregation [3] and microtubule depolymerization [13]. Toxin A also induces a massive colonic epithelial detachment that is associated with a loss of cell-matrix adhesion; this is considered to be a cause of the acute colonic inflammation induced by toxin A [7]. Host factors also appear to be more important than bacterial virulence factors here, as the main mechanisms for C. difficile-mediated gut inflammation appear to be the failure to mount an effective antibody response to C. difficile toxins [5].
When the host immune system is able to produce antibodies against the toxins of C. difficile, the disease severity is reduced; thus, the antibody response is considered to be a major determinant of the disease outcome [10,21]. Tests of serum samples isolated from patients with C. difficile-associated diarrhea revealed antibody responses to toxins A and B [10,21]. Moreover, there was a significant correlation between clinical recovery and high IgG titers against toxin B, suggesting that the antitoxin response of serum IgG can ameliorate C. difficile-induced gut inflammation. Kyne et al. [11] found that patients with high serum IgG became asymptomatic carriers, whereas patients who lacked active immunity suffered from colitis. The antitoxin activity of serum samples isolated in many animal studies has been estimated only by the cell rounding test [2,20]. However, previous reports have indicated that toxins A and B can modulate various signals in colonic epithelial cells, triggering apoptosis [7], cytoskeletal disaggregation [13], massive cell detachment [7], tight junction loss [17], and gut epithelial cell barrier dysfunction in addition to cell rounding [18]. Thus, these additional antitoxin effects of serum should be tested in detail.
Here, we investigated whether antiserum isolated from an antibiotic treatment/C. difficile infection-induced mouse model of gut inflammation could prevent toxin-A-induced apoptosis, cytoskeletal disaggregation, cell detachment, and tight junction loss.
Materials and Methods
Preparation of Vegetative C. difficile and Toxin A
C. difficile strain VPI 10463 (ATCC 43255; ATCC, USA) was cultured in Brain Heart Infusion (BHI) broth (Becton-Dickinson, Franklin Lakes, NY, USA) or BHI broth supplemented with 1.5% agar at 37℃ under anaerobic conditions in polyvinyl incubation bags containing an oxygen-binding system (Anaerocult A; Merck, Germany). Anaerocult A quickly and completely binds oxygen, creating an oxygen-free CO2 atmosphere. An overnight culture of C. difficile grown anaerobically was used to produce vegetative cell suspensions [6]. For oral gavage infection of mice, cells were harvested by centrifugation at 1,500 ×g f or 1 0 min a nd rinsed twice with BHI medium, and the exact cell density of C. difficile was determined using the dilution plate method (1,000-fold dilution). Toxin A was purified from C. difficile as described previously [6]. The purity of this native toxin A was assessed by gel electrophoresis, which confirmed a single protein with the expected molecular mass of 307 kDa.
Induction of Mouse Enteritis Using Toxin A, and Assessment of Inflammation
All mouse studies were approved by the Animal Care and Use Committee of Daejin University (Pocheon, Korea). Male CD1 mice (Daehan Biolink, Korea) weighing 30-35 g were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg). Ileal loops (2 cm) were prepared and injected with control buffer (PBS), toxin A (3 µg), antiserum (10 µl), or toxin A plus antiserum in a volume of 100 µl of PBS. After 4 h, the ileal tissues were removed, opened longitudinally, and washed with PBS. Full-thickness sections were formalin fixed, paraffin embedded, and stained with H&E. Since C. difficile infection was previously reported to stimulate production of the proinflammatory cytokine, tumor necrosis factor-alpha (TNF-α), supernatants were collected from ileal tissues and mouse TNF-α was measured with an enzymelinked immunosorbent assay (ELISA) kit (R&D Systems, USA) [4].
Antibiotic Treatment/C. difficile Infection-Induced Mouse Colitis and Preparation of the Antiserum
The mouse model of antibiotic-induced C difficile-associated disease was developed as reported previously [1]. Male CD1 mice were given antibiotic-containing drinking water (kanamycin, 0.4mg/ml; gentamicin, 0.035mg/ml: colistin, 850 U/ml; metronidazole, 0.215 mg/ml; and vancomycin, 0.045 mg/ml) for 3 days. Thereafter, the mice were given regular autoclaved water for 1 day, and then each received a single intraperitoneal dose of clindamycin (10 mg/kg). One day later, the mice were infected by oral gavage with 0.5 ml of C. difficile strain VPI10463 (5 × 108 CFU/ml). At day 25 postinfection, blood (300 µl) was withdrawn from the retroorbital sinus of each surviving mouse. The isolated blood was left to clot overnight at 4℃, and serum was obtained by centrifugation [1].
Antitoxin Serum Assay
The serum titer was estimated using ELISA, as described previously [20]. Briefly, 100 µl of C. difficile toxin A (30 µg of protein/100 µl) was put in each well of the microtiter ELISA plate and incubated overnight at room temperature. Thereafter, 100 µl of serum diluted 1:20 in PBST containing 1% bovine serum albumin (BSA) was added to each well, and the plates were incubated for 2 h at room temperature. Antitoxin A (goat anti-toxin A; Techlab Inc., Blacksburg, VA, USA) was used as a positive control. For visualization of results, horseradish peroxidase-labeled anti-mouse IgG (Sigma, USA) was added, the plates were incubated for 2 h, O-dianisidine was added as the substrate, and the optical density of the reaction was determined by spectrophotometry at 405 nm (Model 3550; USA).
Cell Culture
HT29 cells (derived from human colorectal adenocarcinoma) were cultured in McCoy’s 5A medium (Invitrogen, Carlsbad, CA, USA) in a 37℃ humidified incubator with a 5% CO2 atmosphere [8].
Antibodies and Reagents
Polyclonal antibodies against p21(WAF1/CIP1), phospho-EpoR, and acetylated tubulin were obtained from Santa Cruz Biotechnology (USA). Polyclonal antibodies against phospho-paxillin, phospho-p53, and caspase-3 were obtained from Cell Signaling Technology (USA). The β-actin antibody and propidium iodide (PI) were purchased from Sigma Aldrich (USA).
Immunoblot Analysis
Colonocytes were washed with cold PBS and lysed in lysis buffer (150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 5 mM EDTA, 1% Nonidet P-40). Equal amounts of protein were fractionated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Antigen-antibody complexes were detected with the LumiGlo reagent (USA).
Antibacterial Activity Assay
The antibacterial activity of the antiserum toward C. difficile was determined as previously described [4]. Briefly, C. difficile (2 × 104 CFU per well in a 96-well plate) was treated with the antiserum (10 µl/well) and incubated anaerobically for 48 h at 37℃. The antibacterial activity was determined by visual inspection and spectrophotometry at 600 nm. In a separate set of experiments, C. difficile was exposed to the antiserum or control mouse serum for 2 days, plated on BHI agar, and then assessed for antibacterial activity [4].
Cell Viability
HT29 cells (3 × 103 cells/well) were treated with medium, control mouse serum, antiserum, toxin A plus control mouse serum, or toxin A plus antiserum for 24 h, and then incubated with MTT dye for 2 h. The solubilization reagent was added, and absorbance was determined by spectrophotometry at 570 nm [13].
Terminal Deoxynucleotidyl Transferase-Mediated Deoxyuridine Triphosphate Nick-End Labeling (TUNEL) Assay
HT29 cells (3 × 103 cells/well) were treated with medium, toxin A, antiserum, or toxin A plus antiserum for 48 h, and then fixed with 4% paraformaldehyde for 20 min. Cells with fragmented nuclear DNA were detected by TUNEL assay (Promega, USA) according to the manufacturer’s instructions, and analyzed using an Eclipse E600 epifluorescence microscope [13].
Statistical Analysis
The results are presented as the mean ± SEM. Data were analyzed using the SIGMA-STAT software package (Jandel Scientific Software, USA). Analyses of variance with protected t-tests were used for intergroup comparisons.
Results and Discussion
Antiserum Isolated from C. difficile-Infected Mice Shows Antitoxin Responses
To explore the inhibitory effect of the antiserum on C. difficile toxin-A-induced apoptosis and cell stress signaling, serum samples were isolated from mice subjected to the mouse gut inflammation model of antibiotic/C. difficile infection-associated disease developed by Chen et al. [1]. The induction of this disease is illustrated in Fig. 1A. Briefly, male mice were given antibiotic (kanamycin, gentamicin, colistin, metronidazole, and vancomycin)-containing drinking water for 3 days, regular autoclaved water for 1 day, and then a single intraperitoneal dose of clindamycin (10 mg/kg). After an additional day, the mice were infected by oral gavage with a suspension of C. difficile (5 × 108 CFU/ml). Twenty-five days post-infection, blood (antiserum) was isolated (Fig. 1A). Both the antiserum and a goat anti-toxin A antibody (positive control) selectively detected immunoblotted toxin A, whereas the unchallenged mouse serum did not (Fig. 1B). ELISA of the serum antitoxin titer showed that C. difficile-challenged mice developed a significant antitoxin IgG response, whereas unchallenged control mice did not (Fig. 1C). Thus, consistent with the previous report that antibiotic treatment plus C. difficile infection of hamsters triggered a serum antitoxin response [2], we herein found that antibiotics treatment plus C. difficile infection of mice for 25 days triggered the production of a high-titer antiserum specific to C. difficile toxin A.
Fig. 1.Induction of mouse colitis using antibiotics plus C. difficile infection, and the antitoxin responses of serum samples. (A) Schematic of the experimental induction of mouse colitis. Blood (antiserum) was isolated from surviving mice (n = 8) on day 25 postinfection. (B) Toxin A was resolved on 5% polyacrylamide gels and probed with goat-toxin A antibody (positive control), control mouse serum (negative control, isolated from unchallenged mice), or antiserum (isolated from mice that survived C. difficile infection following antibiotics treatment). Data are representative of three independent samples. (C) Serum IgG titers of antisera. The bars represent the mean ± SEM of three independent experiments, each with triplicate determinations (n = 8).
The Antiserum Attenuates Toxin-A-Induced Mucosal Damage and Inflammatory Responses
We next investigated whether the antiserum could inhibit toxin-A-induced gut inflammation in mice. Closed ileal loops of CD1 mice were exposed to PBS (100 µl), toxin A (3 µg), antiserum (10 µl), or toxin A plus antiserum for 4 h, and the levels of mucosal damage (i.e., disruption of villi) and TNF-α, two markers of acute inflammation in toxin-A-induced mouse enteritis [13], were measured from tissue samples. As expected, treatment of toxin A (but not the PBS control) caused the marked disruption of villi (Fig. 2A) and increased the production of TNF-α (Fig. 2B). However, both of these toxin-A-induced effects were significantly blocked by co-treatment with the antiserum (Figs. 2A and 2B).
The Antiserum Does Not Show Antimicrobial Activity Against C. difficile
Antibodies produced by immune responses against pathogenic bacteria may show antimicrobial activities in addition to antitoxin responses [2,5,20]. For example, antibacterial and antitoxin responses were observed in serum samples representing clinical cholera caused by Vibrio cholerae O139 [15]. Based on our observation that the antiserum ameliorated toxin-A-induced gut inflammation in our system (Fig. 2), we assessed whether the antiserum could also directly affect the growth of C. difficile (i.e., whether it showed bactericidal activity). C. difficile (2 × 103 to 2 x 105 CFU) was treated with control mouse serum (10 μl) or antiserum (10 μl) and incubated anaerobically at 37℃. After 2 days, bacterial growth was measured by spectrophotometry at 600 nm. Interestingly, the growth rate of C. difficile did not significantly differ in cultures exposed to control mouse serum or the antiserum (Fig. 3A). Inocula treated under the same conditions were also plated on agar, and survival was monitored. Consistent with the above results, the antiserum and control mouse serum both failed to significantly affect the growth rates of C. difficile (Fig. 3B). Our results therefore suggest that the antiserum inhibited the effects of toxin A but had little or no bactericidal activity against C. difficile. We speculate that systemic exposure to toxins A and B may cause strong antitoxin responses through serological immune cell reactions, whereas the C. difficile bacteria themselves are exposed to only a limited mucosal immune response. This hypothesis is consistent with the well-known concept that serum IgG may reflect systemic exposure, whereas serum IgA may be stimulated in response to colonic (rather than systemic) exposure [5].
Fig. 2.Treatment with the antiserum reduces toxin-A-induced gut inflammation in mice. Closed ileal loops were prepared in the distal ilea of CD1 mice (n = 10 mice/group) and treated with PBS, toxin A (3 µg), antiserum (10 µl), or toxin A plus antiserum (all in 100 µl of PBS). After 4 h, animals were sacrificed and ileal loops were removed. (A) Light micrographs of mouse ileum (H&E staining; original magnification, ×200). (B) TNF-α concentrations were measured in the ileal tissues. The bars represent the mean ± SEM of three independent experiments; *, p < 0.005.
Fig. 3.The antiserum does not have bactericidal activity against C. difficile. (A) Antiserum (10 µl) and control mouse serum (con-sera, 10 µl) were mixed with growing cultures of C. difficile (CD, 2 × 104 CFU/ml), and the samples were incubated under anaerobic conditions at 37℃. After 2 days, the antibacterial activity was determined by spectrophotometry at 600 nm. The bars represent the mean ± SEM of three independent experiments. (B) Antiserum (10 µl) and control mouse serum (10 µl) were mixed with growing cultures of C. difficile (CD), the samples were incubated for 2 days at 37℃, and then C. difficile was plated on BHI agar. Data are representative of three independent samples.
The Antiserum Inhibits Toxin-A-Induced Cell Rounding
Just et al. [3] previously reported that C. difficile toxin A causes severe cell rounding via the ability of its glucosyltransferase activity to inactivate Rho and Rac. As shown in Fig. 4, exposure of colonocytes to toxin A for 2 h triggered marked cell rounding, whereas co-treatment with the antiserum significantly blocked this toxin-A-induced cell rounding. These data are consistent with our results showing that the antiserum attenuated toxin-A-induced gut inflammation in the mouse enteritis model (Fig. 2).
Fig. 4.The antiserum blocks toxin A-induced cell rounding. HT29 colonocytes (105 cells/well) were incubated for 2 h with medium (con), toxin A (TxA, 3 nM), control mouse serum (con-sera, 1 µl), antiserum (1 µl), toxin A plus control mouse serum, or toxin A plus antiserum. Light microscopic images are shown (100×). Data are representative of three independent samples.
The Antiserum Inhibits Toxin-A-Induced Apoptosis and Cell Stress Signaling in HT29 Colonocytes
Toxins A and B are known to modulate signaling cascades in gut epithelial cells, triggering (in addition to cell rounding) apoptosis, cytoskeletal disaggregation, massive cell detachment, loss of tight junctions, and gut epithelial cell barrier dysfunction [4,7-9,13,16-18]. To date, however, the neutralizing activities of antibodies against C. difficile toxins have been evaluated solely by measuring the inhibition of toxin-induced cell rounding. Here, we further assessed whether our antiserum could block toxin-Ainduced apoptosis [7,8] and cell stress signaling in colonocytes. As shown in Fig. 5A, toxin A (3 nM) caused a marked loss of cell viability in colonocytes, and this was significantly abrogated by co-treatment with the antiserum (1 μl) but not with control mouse serum. Consistent with our observation that control mouse serum or antiserum alone had no effect on cell rounding (Fig. 4), there was no change in the viability of cultures treated with control mouse serum or the antiserum (Fig. 5A). Regarding apoptosis, TUNEL-based assessment of DNA fragmentation revealed that toxin A treatment induced DNA fragmentation in colonocytes, and this was significantly inhibited by cotreatment with the antiserum (Fig. 5B). The antiserum (but not the control mouse serum) also markedly blocked the toxin-A-induced activation of caspase-3, which is another marker of apoptosis (Fig. 5C).
Fig. 5.The antiserum inhibits toxin-A-induced alterations in HT29 cell signaling. (A) HT29 colonocytes (3 × 103 cells/well) were treated with medium (con), toxin A (TxA, 3 nM), control mouse serum (con-sera, 1 µl), antiserum (1 µl), toxin A plus control mouse serum, or toxin A plus antiserum for 24 h, and cell viability was measured by MTT assay (*, p < 0.005). (B) HT29 cells were treated as described above for 48 h and DNA fragmentation was measured by TUNEL assay. The results shown are representative of three separate experiments. (C) Colonocytes were treated with medium, toxin A, control serum, antiserum, toxin A plus control mouse serum, or toxin A plus antiserum for 48 h. Cell lysates were subjected to 10% polyacrylamide gel electrophoresis and blots were probed with antibodies against caspase-3 and β-actin. (D) The antiserum prevents well-known toxin-A-induced alterations in cell signaling. (E) Closed ileal loops (n = 10 mice/group) were prepared and treated with PBS, toxin A (3 µg), antiserum (10 µl), or toxin A plus antiserum (all in 100 µl of PBS). After 4 h, ileal loops were removed and tissue extracts were subjected to 10% polyacrylamide gel electrophoresis. The blotted membranes were probed with antibodies against caspase-3, acetylated tubulin, or β-actin. The presented results are representative of three independent experiments.
With respect to stress signaling, toxin A treatment of colonocytes is known to inhibit the activating phosphorylation of the cell adhesion component, paxillin; inhibit the activating phosphorylation of the cell adhesion molecule, EPO receptor (EpoR) [14]; inactivate tubulin (deacetylation), which forms microtubules; and activate the programmed cell death activators (phosphorylation), p21(WAF1/CIP1) and p53 [7]. Based on these reports, we assessed whether the antiserum could rescue these toxin-A-induced effects in colonocytes. HT29 cells were incubated with medium, toxin A (3 nM), control mouse serum, antiserum, toxin A plus control mouse serum, or toxin A plus antiserum for 6 h. As expected, toxin A treatment markedly decreased paxillin phosphorylation, EpoR phosphorylation, and tubulin acetylation, but these effects were significantly rescued by co-treatment with the antiserum (Fig. 5D). Marked increases in the phosphorylation of p21(WAF1/CIP1) and p53 were also observed in toxin-A-treated cells, but these effects were significantly reduced by co-treatment with the antiserum (Fig. 5D). Similarly, toxin A exposure markedly increased caspase-3 activation and tubulin deacetylation in mouse ileal loops, but these changes were completely blocked and significantly rescued, respectively, by co-treatment with the antiserum (Fig. 5E).
In conclusion, we herein extend the previous reports of toxin-A-induced cell rounding and antiserum responses against C. difficile infection-mediated gut inflammation. We show that our antiserum ameliorated the severe toxin-Ainduced cell stress signalings that may trigger apoptosis, cytoskeletal disaggregation, cell detachment, and tight junction loss in colonic epithelial cells. Our results collectively indicate that this antitoxin could have biotherapeutic effects against the various cellular toxicities of C. difficile toxins.
Abbreviations
C. difficile, Clostridium difficile, TxA, toxin A; Ab, antibody; TNF-α, tumor necrosis factor-alpha; H&E staining, hematoxylin and eosin staining; MTT, 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling
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