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. 2014 Aug;58(8):4642–4650. doi: 10.1128/AAC.02783-14

Fidaxomicin Inhibits Clostridium difficile Toxin A-Mediated Enteritis in the Mouse Ileum

Hon Wai Koon a, Samantha Ho a, Tressia C Hing a, Michelle Cheng a, Xinhua Chen b, Yoshi Ichikawa c, Ciarán P Kelly b, Charalabos Pothoulakis a,✉
PMCID: PMC4135997  PMID: 24890583

Abstract

Clostridium difficile infection (CDI) is a common, debilitating infection with high morbidity and mortality. C. difficile causes diarrhea and intestinal inflammation by releasing two toxins, toxin A and toxin B. The macrolide antibiotic fidaxomicin was recently shown to be effective in treating CDI, and its beneficial effect was associated with fewer recurrent infections in CDI patients. Since other macrolides possess anti-inflammatory properties, we examined the possibility that fidaxomicin alters C. difficile toxin A-induced ileal inflammation in mice. The ileal loops of anesthetized mice were injected with fidaxomicin (5, 10, or 20 μM), and after 30 min, the loops were injected with purified C. difficile toxin A or phosphate-buffered saline alone. Four hours after toxin A administration, ileal tissues were processed for histological evaluation (epithelial cell damage, neutrophil infiltration, congestion, and edema) and cytokine measurements. C. difficile toxin A caused histologic damage, evidenced by increased mean histologic score and ileal interleukin-1β (IL-1β) protein and mRNA expression. Treatment with fidaxomicin (20 μM) or its primary metabolite, OP-1118 (120 μM), significantly inhibited toxin A-mediated histologic damage and reduced the mean histology score and ileal IL-1β protein and mRNA expression. Both fidaxomicin and OP-1118 reduced toxin A-induced cell rounding in human colonic CCD-18Co fibroblasts. Treatment of ileal loops with vancomycin (20 μM) and metronidazole (20 μM) did not alter toxin A-induced histologic damage and IL-1β protein expression. In addition to its well known antibacterial effects against C. difficile, fidaxomicin may possess anti-inflammatory activity directed against the intestinal effects of C. difficile toxins.

INTRODUCTION

Clostridium difficile is a common infection that is associated with diarrhea, disrupted gut function, and increasing morbidity and mortality (1, 2). C. difficile produces two toxins, toxin A and toxin B, which trigger intestinal inflammation and diarrhea in animals and humans (1, 2). Generally, C. difficile infection (CDI) is treated by the administration of antibiotics, including metronidazole or vancomycin (3); however, these drugs are frequently associated with recurrent CDI (4). Recently, two large double-blind phase III trials showed that the antibiotic fidaxomicin was noninferior to vancomycin treatment regarding clinical cure rates and was associated with substantially lower rates of recurrent CDI (5–7). Several mechanisms may mediate the beneficial effects of fidaxomicin in CDI, including antimicrobial activity against C. difficile strains (8–10), which is due to inhibition of the transcription of bacterial RNA by RNA polymerases (11), and reduction of toxin A and B production by C. difficile (12).

Fidaxomicin is the first in a new class of 18-membered antibacterial macrolides (13). It has been reported that several 14- or 15-membered antibiotic macrolides, such as clarithromycin and azithromycin, which inhibit bacterial ribosome activity, possess anti-inflammatory effects (14, 15). Other, nonantibiotic macrolides, including tacrolimus and sirolimus, are used predominantly as immunomodulators. Based on this consideration and on the ability of C. difficile toxins to mediate CDI and cause an in vivo inflammatory response in animal models, we examined the hypothesis that fidaxomicin possesses anti-inflammatory effects in C. difficile toxin A-mediated enteritis in vivo. To test this hypothesis, we used the well-established mouse C. difficile toxin A ileal loop model and examined the ability of fidaxomicin and its active metabolite OP-1118 (60 or 120 μM) to modulate intestinal inflammation and histologic damage in response to ileal C. difficile toxin A administration. The ability of vancomycin and metronidazole to modulate toxin A-associated intestinal inflammation in this model was also evaluated.

MATERIALS AND METHODS

C. difficile culture and toxin purification.

C. difficile strain VPI 10463 (ATCC stock 43255) was cultured in Difco cooked meat medium (BD number 226730; Fisher Scientific) at 37°C under anaerobic conditions, and toxin A was purified to homogeneity as previously described (16). The cytotoxicity of toxin A was determined by cell rounding as previously described (16).

Ileal loop mouse studies.

Ten-week-old male C57BL/6 mice were purchased from Jackson Laboratories and maintained at the University of California Los Angeles (UCLA) animal facility under standard conditions. Mice received standard pelleted chow and tap water ad libitum. Mice were anesthetized with isoflurane, and 2-cm ileal loops were formed (one loop per animal) by tying the ileum up with surgical sutures. The ileal loops of the anesthetized mice were injected with fidaxomicin (5, 10, or 20 μM), OP-1118 (60 or 120 μM), metronidazole (20 μM), vancomycin (20 μM), or vehicle (dimethyl sulfoxide [DMSO]). After 30 min, the loops were injected with purified C. difficile toxin A (10 μg in 50 μl phosphate-buffered saline [PBS]) or PBS alone in a 200-μl volume (n = 6 mice per group) as we previously described (16). The final concentration of DMSO in the ileal loop was 0.8%. The abdomen was sealed with surgical sutures and wound clips, and the mice were returned to consciousness. After 4 h, the ileal tissues were processed for histological evaluation (epithelial cell damage, neutrophil infiltration, congestion, and edema) and cytokine measurements (16). The animal studies were approved by the Institutional Animal Research Committee of UCLA.

Histology scoring.

The ileal tissues of the mice were sectioned, stained with hematoxylin and eosin (H&E), and analyzed by two independent observers in a blinded manner. The severity of enteritis and colitis was graded using 3 previously published parameters, (i) epithelial tissue damage, (ii) hemorrhagic congestion and mucosal edema, and (iii) neutrophil infiltration (17). A score of 0 to 3 was assigned to each parameter. The total histology score was determined by the sum of the three parameter scores (0 to 9). The histological score was calculated by observing at least 20 different fields of H&E-stained ileal sections from each group at ×100 magnification.

IL-1β ELISA.

The levels of the proinflammatory mediator mouse interleukin-1β (IL-1β) (catalog number DY401; R&D Systems, Minneapolis, MN) were measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturers' instructions.

Quantitative real-time reverse transcription-PCR.

Total RNA was isolated by using an RNeasy kit (catalog number 74106; Qiagen, Valencia, CA) and reverse transcribed into cDNA using a Superscript III kit (catalog number 11752; Invitrogen, Carlsbad, CA). Quantitative PCRs were run in an ABI Prism 7700 fast sequence detector system as previously described (16). The levels of mRNA were determined by using cataloged primers (Invitrogen) for mouse IL-1β (Mm00434228_m1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Mm99999915_g1). The results were expressed as the relative fold difference from the results for controls.

Cell rounding experiments.

Human colonic CCD-18Co fibroblasts were cultured in minimal essential medium with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (105 cells/well) in 12-well plates. Cells were grown in 1 ml medium per well to around 80% confluence. Cells were serum starved overnight and then incubated with PBS containing 0.8% DMSO (vehicle) or vehicle containing 20 μM fidaxomicin or 120 μM OP-1118 for 30 min, followed by the addition of 0.1 ng/ml C. difficile toxin A for 6 h at 37°C. For all groups, the final concentration of DMSO was 0.8%. The volume of DMSO, fidaxomicin, OP-1118, vancomycin, or metronidazole added to the culture was 8 μl/well. At the end, microphotographs to observe cell rounding were taken in a blinded manner.

Immunohistochemistry.

The ileal tissues were fixed in 4% paraformaldehyde and embedded in paraffin. After incubation with blocking buffer, sections were incubated with a rabbit polyclonal anti-phosphorylated-extracellular signal-regulated kinase 1 and 2 (phospho-ERK1/2) antibody (product number 4370, 1:50 dilution; Cell Signaling, Danvers, MA, USA) overnight at 4°C. After washing, sections were incubated with bovine anti-rabbit IgG and slides were stained with an ABC kit for color development (sc-2018; Santa Cruz). Images were taken with a Zeiss AX10 microscope at magnification of ×200 in a blinded manner. Assistance with the H&E staining and immunohistochemistry experiments was provided by the histology core facility of the University of California Los Angeles.

Statistical analyses.

Quantitative results were expressed as means with standard errors of the means. The results were analyzed using Prism professional statistics software (Graphpad, San Diego, CA). Student's t tests with Mann-Whitney posttests were used for intergroup comparisons.

RESULTS

Fidaxomicin, but not metronidazole and vancomycin, inhibits C. difficile toxin A-mediated enteritis in mice.

Although fidaxomicin, metronidazole, and vancomycin are known to possess potent antimicrobial effects against C. difficile (18), the potential anti-inflammatory effects of these drugs are not known. The C. difficile toxin A ileal loop enteritis model can induce enteritis without the involvement of the C. difficile bacterium and can be used to study anti-inflammatory effects of antibacterial agents (16). As shown by the results in Fig. 1A, exposure of the mouse ileum to C. difficile toxin A (10 μg per ileal loop) resulted in significant tissue damage after 4 h compared to the results for normal controls. The histologic changes included substantial epithelial damage and neutrophil infiltration with congestion and edema, consistent with prior reports (16, 17). The histologic changes are also reflected by a significantly increased mean histology score compared to that of the control group (Fig. 1B). Pretreatment with fidaxomicin (5 to 20 μM) significantly reduced the toxin A-induced histology damage and associated histology score, suggesting anti-inflammatory effects (Fig. 1A and B).

FIG 1.

FIG 1

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Fidaxomicin inhibits C. difficile toxin A-mediated histological damage in the ileum. (A) Ileal loops of mice were pretreated with fidaxomicin (5 to 20 μM) or 0.8% DMSO followed by toxin A (10 μg per ileal loop) or PBS alone (200 μl). The ileal loops were obtained 4 h later for H&E staining. Toxin A caused destruction of villous structure that was reduced by fidaxomicin (arrows). (B) Histology scores were evaluated as described in Materials and Methods. C. difficile toxin A significantly increased the mean histology score compared to that of the normal control group. Fidaxomicin at 5 to 20 μM dose-dependently reduced the mean histology score in C. difficile toxin A-treated ileal loops. n = 6 mice per group.

On the other hand, pretreatment with metronidazole (20 μM) or vancomycin (20 μM) did not significantly alter toxin A-mediated histology damage (Fig. 2A and B), suggesting that these two antibiotics do not exert anti-inflammatory effects against C. difficile toxin A in vivo.

FIG 2.

FIG 2

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Metronidazole and vancomycin do not affect C. difficile toxin A-mediated histological damage in mouse ileum. (A) Ileal loops of mice were pretreated with metronidazole (20 μM), vancomycin (20 μM), or DMSO (0.8%) followed by the administration of C. difficile toxin A (10 μg per ileal loop) or PBS alone (200 μl). After 4 h, ileal loops were processed for H&E staining. Toxin A caused destruction of villous structure that was not affected by metronidazole or vancomycin (arrows). (B) Histology scores were evaluated as described in Materials and Methods. Neither metronidazole nor vancomycin altered the score for histological damage in C. difficile toxin A-treated ileal loops. n = 6 mice per group.

Fidaxomicin, but not metronidazole and vancomycin, inhibits toxin A-mediated IL-1β expression in the ileum.

C. difficile toxin A increases the transcription of the proinflammatory cytokine IL-1β in the human colon (19), and this cytokine is elevated in patients with C. difficile colitis (20). Ileal administration of toxin A significantly increased ileal colonic IL-1β protein and mRNA expression in mice (Fig. 3A and B). Pretreatment of ileal loops with 20 μM fidaxomicin, but not 5 or 10 μM, significantly reduced C. difficile toxin A-induced IL-1β protein levels (Fig. 3A), while all concentrations of fidaxomicin (5 to 20 μM) almost abolished toxin A-induced IL-1β mRNA expression (Fig. 3B). Moreover, pretreatment with metronidazole or vancomycin did not affect toxin A-induced IL-1β expression in the mouse ileum under the same experimental conditions (Fig. 3C).

FIG 3.

FIG 3

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Fidaxomicin reduces C. difficile toxin A-induced ileal IL-1β expression, but metronidazole and vancomycin do not. (A) C. difficile toxin A (10 μg per ileal loop) significantly induced ileal IL-1β protein expression (P = 0.0003), while fidaxomicin (20 μM) significantly reduced C. difficile toxin A-induced ileal IL-1β protein expression (P = 0.0004). (B) C. difficile toxin A (10 μg per ileal loop) significantly induced ileal IL-1β mRNA expression, (P = 0.0101) while fidaxomicin significantly reduced C. difficile toxin A-induced ileal IL-1β mRNA expression (5 μM, P = 0.0351; 10 μM, P = 0.0096; 20 μM, P = 0.0384). Fidaxomicin also reduced basal ileal IL-1β protein (P = 0.0062) but not mRNA expression. (C) C. difficile toxin A (10 μg per ileal loop) significantly induced ileal IL-1β protein expression (P = 0.0036). Metronidazole and vancomycin (20 μM) did not alter C. difficile toxin A-induced ileal IL-1β protein levels. n = 6 mice per group.

OP-1118 reduces C. difficile toxin A-mediated tissue damage and IL-1β expression in mouse ileum.

To confirm and extend our results on fidaxomicin in toxin A-induced intestinal inflammation, we examined the effect of its primary metabolite, OP-1118, on this in vivo toxin A response. OP-1118 at 120 μM significantly reduced C. difficile toxin A-mediated ileal damage, as shown by the reduced histology score (Fig. 4A and B). Similar to fidaxomicin, OP-1118 at 120 μM but not at 60 μM significantly reduced C. difficile toxin A-associated IL-1β protein and/or mRNA expression in the mouse ileum (Fig. 5A and B).

FIG 4.

FIG 4

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OP-1118 has anti-inflammatory effects against C. difficile toxin A in ileum. (A) Ileal loops of mice were pretreated with OP-1118 (60 to 120 μM) or DMSO (0.8%) followed by C. difficile toxin A (10 μg per ileal loop) or PBS alone (200 μl). Ileal loops were processed after 4 h for H&E staining. Toxin A caused destruction of villous structure that was reduced by OP-1118 (arrows). (B) Histology scores were evaluated as described in Materials and Methods. C. difficile toxin A significantly increased the mean histology score (P = 0.0001) compared to that of the normal control group. OP-1118 (120 μM) significantly reduced the mean histology score in toxin A-treated ileal loops (P = 0.0004). n = 6 mice per group. n.s., not significant.

FIG 5.

FIG 5

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OP-1118 inhibits C. difficile toxin A-induced IL-1β expression in ileum. (A) C. difficile toxin A (10 μg per ileal loop) significantly induced IL-1β protein expression (P = 0.0001) in ileal loops that was significantly reduced by OP-1118 (120 μM) (P = 0.0286). (B) C. difficile toxin A significantly induced IL-1β mRNA expression (P = 0.0101) in ileal loops that was significantly reduced by OP-1118 (120 μM) (P = 0.0127). OP-1118 also reduced basal ileal IL-1β protein (P = 0.0402) but not mRNA expression. n = 6 mice per group.

Fidaxomicin reduces C. difficile toxin A-mediated MAP kinase phosphorylation in mouse ileum.

C. difficile toxin A activates mitogen-activated protein (MAP) kinases, including ERK1/2, in vivo and in vitro (21, 22). Here, we observed induction of ERK phosphorylation in ileal loops exposed to C. difficile toxin A (Fig. 6A), while the administration of fidaxomicin substantially diminished this response. In contrast, metronidazole and vancomycin did not significantly alter toxin A-mediated ERK1/2 phosphorylation in the mouse ileum (Fig. 6B).

FIG 6.

FIG 6

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Fidaxomicin and OP-1118 inhibit C. difficile toxin A-mediated ERK phosphorylation in ileum. Ileal loops of mice were pretreated with fidaxomicin (20 μM), OP-1118 (120 μM), metronidazole (20 μM), vancomycin (20 μM), or DMSO (0.8%) followed by C. difficile toxin A (10 μg per ileal loop) or PBS alone (200 μl). After 4 h, ileal loops were processed for phospho-ERK immunohistochemistry as described in Materials and Methods. (A) C. difficile toxin A induced ERK phosphorylation in ileal mucosal tissues that was diminished by fidaxomicin or OP-1118 treatment. (B) Metronidazole and vancomycin treatment had no effect on C. difficile toxin A-induced ERK phosphorylation in ileal tissues. n = 6 mice per group.

Fidaxomicin and OP-1118 reduce C. difficile toxin A-mediated cell rounding in human colonic fibroblasts.

To understand the protective mechanism of fidaxomicin and OP-1118, we examined their ability to affect toxin A-associated cell rounding using human colonic CCD-18Co fibroblasts. Exposure of CCD-18Co fibroblasts to toxin A for 6 h resulted in cell rounding (Fig. 7). Coincubation of cells with fidaxomicin at 20 μM or OP-1118 at 120 μM partially reduced the cell rounding effect of toxin A (Fig. 7A). Vancomycin and metronidazole did not prevent toxin A-induced cell rounding (Fig. 7B). Similar results were obtained when fibroblastlike mouse 3T3-L1 preadipocytes were used (data not shown). Together, these results indicate that fidaxomicin and OP-1118 protect cells against toxin A-mediated cytoskeletal damage.

FIG 7.

FIG 7

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Fidaxomicin and OP-1118 prevented C. difficile toxin A-mediated cell rounding. (A) Serum-starved CCD-18Co human colonic fibroblasts were treated with DMSO (0.8%), C. difficile toxin A (0.1 ng/ml), fidaxomicin (20 μM), or OP-1118 (120 μM) for 6 h. The spindle shape of fibroblasts was lost after exposure to toxin A, but this change was prevented by coincubation with fidaxomicin or OP-1118. (B) Serum-starved CCD-18Co fibroblasts were treated with DMSO (0.8%), C. difficile toxin A (0.1 ng/ml), metronidazole (20 μM), or vancomycin (20 μM) for 6 h. Metronidazole and vancomycin failed to protect cells from toxin A-induced cell rounding. Results shown are representative of 2 independent experiments.

DISCUSSION

C. difficile mediates CDI and intestinal inflammation by a mechanism involving the release of two potent exotoxins, toxin A and toxin B (1, 23). Fidaxomicin is a new antibiotic member of the macrolide family (24), recently approved by the FDA for use against CDI (25). Although its efficacy is similar to that of vancomycin, the use of fidaxomicin is associated with fewer recurrent episodes of CDI (6). The mechanisms involved in this response are still under investigation, but the reduced rates of recurrent CDI following fidaxomicin administration may be related to the preservation of commensal microflora as compared to the effect of vancomycin (26, 27), inhibition of sporulation (28), or an inhibitory effect on toxin production by C. difficile (12).

The in vivo mechanisms by which C. difficile toxins A and B mediate diarrhea and inflammation have been elucidated in large part by studies with relevant experimental models, including the ileal loop model of toxin A-induced enteritis (29–31). The beneficial effects of fidaxomicin in CDI and the ability of other macrolides to possess anti-inflammatory responses in nongastrointestinal organs (14, 15) led us to hypothesize that fidaxomicin may affect inflammatory responses against C. difficile toxin A by modulating signaling pathways that regulate the mucosal inflammation activated by this toxin. We were unable to test the ability of these drugs to inhibit the effects of toxin B in ileal loops in vivo, since the mouse intestine is insensitive to this toxin in this experimental system (32). Using this model and purified toxin A, we show here that fidaxomicin significantly reduced C. difficile toxin A-mediated histological damage (Fig. 1), IL-1β expression (Fig. 3), and ERK phosphorylation (Fig. 6) in the mouse ileum. We also show that the primary metabolite of fidaxomicin, OP-1118 (33), can also inhibit C. difficile toxin A-mediated inflammatory responses in the mouse ileum similarly to its parent compound (Fig. 4 and 5). Thus, OP-1118 may at least partially mediate the anti-inflammatory effects of fidaxomicin against C. difficile toxin A in the intestine. Our results indicate that pretreatment with metronidazole and vancomycin, commonly used for therapy of CDI, did not significantly alter the histologic damage, IL-1β expression, or ERK activation in response to toxin A in vivo and did not affect cell rounding in response to this toxin in vitro. On the other hand, both metronidazole and vancomycin have been shown to possess anti-inflammatory effects under different in vivo and in vitro conditions (34–37). The different inflammatory stimuli (Staphylococcus aureus toxin or lipopolysaccharide) used in the studies mentioned above may account for the inability of metronidazole and vancomycin to alter C. difficile toxin A-associated responses that was shown in our study.

Our results demonstrating that fidaxomicin and OP-1118 reduce ERK activation in response to toxin A in vivo suggest that this pathway may be important to the anti-inflammatory actions of this macrolide during toxin A enteritis (Fig. 6). ERK phosphorylation is required to elicit the secretion of proinflammatory cytokines in response to C. difficile toxins in vivo and in vitro (21, 22, 38). Interestingly, another macrolide, azithromycin, suppresses IL-1β production and ERK phosphorylation in human peripheral blood mononuclear cells (39), while azithromycin and clarithromycin modulate proinflammatory cytokine secretion in human bronchial epithelial cells, in part through ERK activation (40). Thus, inhibition of proinflammatory cytokine transcription and MAP kinase activation may represent common anti-inflammatory responses of several macrolides (41), including fidaxomicin. This may explain why these two drugs can preserve the normal functions of cells exposed to C. difficile toxin A. The possibility, however, that the protective effects of fidaxomicin are involved in the lower rates of recurrence of CDI following fidaxomicin treatment remains to be investigated.

Our results also indicate that both fidaxomicin and OP-1118, but not vancomycin or metronidazole, reduce the cytopathic effects of C. difficile toxin A in colonic CCD-18Co fibroblasts (Fig. 7), suggesting that fidaxomicin and OP-1118 may interfere with the mechanisms involved in this response. The primary molecular mechanism by which C. difficile toxins mediate actin disaggregation and cell rounding following toxin cell surface binding and internalization is glucosylation of Rho, Rac, and cdc42 at threonine 37, leading to the inactivation of these small GTP binding proteins and, eventually, to cell rounding (42). Further exploration of the specific mechanisms contributing to the inhibitory effects of fidaxomicin and OP-1118 on toxin A-associated cytoskeletal effects is warranted.

In summary, fidaxomicin significantly reduces C. difficile toxin A-mediated proinflammatory cytokine expression, ileal tissue damage, and ERK activation in mouse intestinal mucosa. These results strongly suggest that fidaxomicin, like other macrolides, possesses anti-inflammatory activities against C. difficile toxin A that are independent of its well established antimicrobial effects.

ACKNOWLEDGMENTS

We thank Yuzu Kubota, Deanna Tran, and Irene Chang for their assistance with our experiments.

This study was supported by Cubist Pharmaceuticals (to C.P.). H.W.K. was supported by a Crohn's and Colitis Foundation of America Career Development Award (number 2691) and NIH grant K01 DK084256. S.H. was supported by a Crohn's and Colitis Foundation of America student research fellowship (number 3831). M.C. was supported by a Crohn's and Colitis Foundation of America student research fellowship (number 287244). Support was also provided by the Blinder Research Foundation for Crohn's Disease (C.P.) and the Eli and Edythe Broad Chair (C.P.). C.P.K. is supported by NIH grant RO1 AI095256, and X.C. by a Career Development Award from the Crohn's and Colitis Foundation of America.

Footnotes

Published ahead of print 2 June 2014

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