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Fidaxomicin inhibits Clostridium difficile toxin A – mediated enteritis in mouse ileum. 1

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Hon Wai Koon1, Samantha Ho1, Tressia C. Hing1, Michelle Cheng1, Xinhua Chen2, Yoshi Ichikawa3, 3

Ciarán P. Kelly2, and Charalabos Pothoulakis1. 4

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Author affiliation: 6

1 Center for Inflammatory Bowel Diseases, Division of Digestive Diseases, David Geffen School of 7

Medicine at the University of California Los Angeles, Los Angeles, CA 90095, USA. 8

2 Division of Gastroenterology, Department of Medicine, Beth Israel Deaconess Medical Center, 9

Harvard Medical School, Boston, MA 02215, USA. 10

3 Cubist Pharmaceuticals, Inc., 65 Hayden Avenue, Lexington, MA 02421 11

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Running title: Anti-inflammatory effects of fidaxomicin 13

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Corresponding author: 15

Charalabos Pothoulakis, M.D., 16

Center for Inflammatory Bowel Diseases, 17

Division of Digestive Diseases, 18

David Geffen School of Medicine, 19

MRL Building, Room 1240 20

675 Charles E. Young Dr. South, Los Angeles, CA 90095 21

Office phone: 310-825-9104, Fax: 310-825-3542, e-mail: cpothoulakis@mednet.ucla.edu 22

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AAC Accepts, published online ahead of print on 2 June 2014Antimicrob. Agents Chemother. doi:10.1128/AAC.02783-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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Abstract: 25

Clostridium difficile infection (CDI) is a common, debilitating infection with high morbidity 26

and mortality. C. difficile causes diarrhea and intestinal inflammation by releasing two toxins, toxin A 27

and toxin B. The macrolide antibiotic fidaxomicin was recently shown to be effective in treating CDI 28

and its beneficial effect was associated with fewer recurrences in CDI patients. Since other macrolides 29

possess anti-inflammatory properties, we examined the possibility that fidaxomicin alters C. difficile 30

toxin A-induced ileal inflammation in mice. Ileal loops of anesthetized mice were injected with 31

fidaxomicin (5, 10 or 20 μM) and after 30 minutes, loops were injected with purified C. difficile toxin 32

A or PBS alone. Four hours after toxin A administration, ileal tissues were processed for histological 33

evaluation (epithelial cell damage, neutrophil infiltration, congestion and edema) and cytokine 34

measurements. C. difficile toxin A caused histologic damage evident by increased histologic score and 35

ileal IL-1β protein and mRNA expression. Treatment with fidaxomicin (20 μM) or its primary 36

metabolite, OP-1118 (120 μM), significantly inhibited toxin A- mediated histologic damage and 37

histology score, and reduced ileal IL-1β protein and mRNA expression. Both fidaxomicin and OP-38

1118 reduced toxin A induced cell rounding in human colonic CCD-18Co fibroblasts. Treatment of 39

ileal loops with vancomycin (20 μM) and metronidazole (20 μM) did not alter toxin A-induced 40

histologic damage and IL-1β protein expression. In addition to its well known antibacterial effects 41

against C. difficile, fidaxomicin may possess anti-inflammatory activity directed against the intestinal 42

effects of C. difficile toxins. 43

44

45

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Introduction: 46

C. difficile is a common infection associated with diarrhea, disrupted gut function and 47

increasing morbidity and mortality (1, 2). C. difficile produces two toxins, toxin A and toxin B, which 48

trigger intestinal inflammation and diarrhea in animals and humans (1, 2). Generally, C. difficile 49

infection (CDI) is treated by administration of antibiotics including, metronidazole or vancomycin (3) 50

which, however, are frequently associated with recurrent CDI (4). Recently, two large, double-blind 51

phase III trials showed that the antibiotic fidaxomicin was noninferior to vancomycin treatment 52

regarding clinical cure rates and was associated with substantially lower recurrent CDI (5-7). Several 53

mechanisms may mediate the beneficial effects of fidaxomicin in CDI, including antimicrobial activity 54

against C. difficile strains (8-10) by inhibiting transcription of bacterial RNA by RNA polymerases 55

(11) and reduction of toxin A and B production by C. difficile (12). 56

57

Fidaxomicin is a new class of 18-membered antibacterial macrolide (13). It has been reported 58

that several 14 or 15-membered antibiotic macrolides, such as clarithromycin and azithromycin that 59

inhibit bacterial ribosome activity, possess anti-inflammatory effects (14, 15). Other, non-antibiotic, 60

macrolides including tacrolimus and sirolimus are used predominantly as immunomodulators. Based 61

on this consideration and on the ability of C. difficile toxins to mediate CDI and cause an in vivo 62

inflammatory response in animal models, we examined the hypothesis that fidaxomicin possesses anti-63

inflammatory effects in C. difficile toxin A mediated enteritis in vivo. To test this hypothesis, we used 64

the well-established mouse C. difficile toxin A ileal loop model and examined the ability of 65

fidaxomicin and its active metabolite OP-1118 (60 or 120 μM) to modulate intestinal inflammation 66

and histologic damage in response to ileal C. difficile toxin A administration. The ability of 67

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vancomycin and metronidazole to modulate toxin A-associated intestinal inflammation in this model 68

was also evaluated. 69

70

Materials and Methods: 71

C. difficile culture and toxin purification: 72

C. difficile strain VPI 10463 (ATCC stock 43255) was cultured in Difco cooked meat media 73

(#226730 BD, Fisher scientific) at 37oC in anaerobic conditions and toxin A was purified to 74

homogeneity as previously reported (16). Cytotoxicity of toxin A was determined by cell rounding as 75

previously described (16). 76

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Ileal Loop Mouse Studies: 78

Male 10 weeks old C57BL/6 mice were purchased from Jackson Laboratories and maintained 79

at the University of California, Los Angeles (UCLA) animal facility under standard conditions. Mice 80

received standard pelleted chow and tap water ad libitum. Mice were anesthetized with isoflurane. Two 81

cm ileal loops were formed (one loop per animal) by tying up with surgical sutures. Ileal loops of 82

anesthetized mice were injected with fidaxomicin (5, 10 or 20 μM), OP-1118 (60 or 120 μM), 83

metronidazole (20 μM), vancomycin (20 μM) or vehicle (DMSO). After 30 minutes, loops were 84

injected with purified C. difficile toxin A (10 μg in 50 µl PBS) or PBS alone in 200 μl volume (n=6 85

mice per group) as we previously described (16). The final concentration of DMSO in the ileal loop is 86

0.8%. The abdomen was sealed by surgical sutures and wound clips and mice were returned to 87

consciousness. After 4 hours, ileal tissues were processed for histological evaluation (epithelial cell 88

damage, neutrophil infiltration, congestion and edema) and cytokine measurements (16). Animal 89

studies were approved by the Institutional Animal Research Committee of UCLA. 90

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Histology scoring: 92

Ileal tissues of mice were sectioned and stained with H&E and analyzed by two independent 93

observers in a blinded manner. Severity of enteritis and colitis was graded using 3 parameters 94

previously published (17): (i) epithelial tissue damage; (ii) hemorrhagic congestion and mucosal 95

edema; (iii) neutrophil infiltration. A score of 0-3 was assigned to each parameter. Total histology 96

score was determined by the sum of all these three parameter scores (0-9). The histological score was 97

calculated by observing at least 20 different fields of H&E-stained ileal sections at 100x from each 98

group. 99

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IL-1β ELISA: 101

The levels of pro-inflammatory mediator mouse interleukin 1 beta (IL-1β) (DY401 R&D 102

Systems, Minneapolis, MN) were measured, according to manufacturers’ instructions. 103

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Quantitative real-time RT-PCR: 105

Total RNA was isolated by RNeasy kit (#74106, Qiagen, CA) and reverse transcribed into 106

cDNA by a Superscript III kit (#11752, Invitrogen, Carlsbad, CA). Quantitative PCR reactions were 107

run in an ABI Prism 7700 Fast sequence detector system as previously described (16). The levels of 108

mRNA were determined by using cataloged primers (Invitrogen) for mouse IL-1β (Mm00434228_m1) 109

and GAPDH (Mm99999915_g1). Results were expressed as relative fold difference. 110

111

Cell rounding experiments: 112

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Human colonic CCD-18Co fibroblasts were cultured in MEM media with 10% FBS and 1% 113

penicillin and streptomycin (105 cells/well) in 12 well plates. Cells were grown in 1ml/well media to 114

around 80% confluence. Cells were serum starved overnight and then incubated with PBS containing 115

0.8% DMSO (vehicle), or vehicle containing fidaxomicin 20 μM or OP-1118 120 μM for 30 minutes, 116

followed by addition of 0.1 ng/ml C. difficile toxin A for 6 hours in 37oC. For all groups, the final 117

concentration of DMSO was 0.8%. The volume of DMSO, fidaxomicin, OP-1118, vancomycin or 118

metronidazole added to culture was 8 μl/well. At the end, microphotographs were taken to observe cell 119

rounding in a “blinded” manner. 120

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Immunohistochemistry: 122

Ileal tissues were fixed in 4% paraformaldehyde and embedded in paraffin. After incubation 123

with blocking buffer, sections were incubated with a rabbit polyclonal anti-phospho-ERK1/2 antibody 124

(#4370, Cell signaling, Danvers, MA, USA, 1:50 dilution) overnight at 4oC. After washing, sections 125

were incubated with bovine anti-rabbit IgG and slides were stained with an ABC kit for color 126

development (Santa Cruz, sc-2018). Images were taken with a Zeiss AX10 microscope at 127

magnification of 200X taken in a “blinded” manner. H&E staining and immunohistochemistry 128

experiments were assisted by the histology core facility of the University of California Los Angeles. 129

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Statistical analyses: 131

Quantitative results were expressed with error bars as mean+/-standard error of the mean. 132

Results were analyzed using Prism professional statistics software program (Graphpad, San Diego, 133

CA). Student’s t-tests with Mann-Whittney post tests were used for intergroup comparisons. 134

135

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Results: 136

Fidaxomicin, but not Metronidazole or Vancomycin, inhibits C. difficile toxin A-mediated 137

enteritis in mice. 138

Although fidaxomicin, metronidazole and vancomycin are known to possess potent 139

antimicrobial effects against C. difficile (18), the potential anti-inflammatory effects of these drugs are 140

not known. The C. difficile toxin A ileal loop enteritis model can induce enteritis without involvement 141

of C. difficile bacterium and can be used to study anti-inflammatory effects of anti-bacterial agents 142

(16). As shown in Figure 1A, exposure of mouse ileum to C. difficile toxin A (10 μg per ileal loop) 143

resulted in significant tissue damage after 4 hours of incubation, compared to normal control. 144

Histologic changes included substantial epithelial damages and neutrophil infiltration with congestion 145

and edema, consistent with prior reports (16, 17). Histologic changes are also reflected by significantly 146

increased histology score compared to control (Figure 1B). Pretreatment with fidaxomicin (5-20 μΜ) 147

significantly reduced the toxin A-induced histology damage and associated histology score, suggesting 148

anti-inflammatory effects (Figure 1A and 1B). 149

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On the other hand, pretreatment with metronidazole (20 μM) or vancomycin (20 μM) did not 151

significantly alter toxin A mediated histology damage (Figure 2A and 2B), suggesting that these two 152

antibiotics do not exert anti-inflammatory effects against C. difficile toxin A in vivo. 153

154

Fidaxomicin, but not metronidazole or vancomycin, inhibits toxin A mediated IL-1β expression 155

in ileum. 156

C. difficile toxin A increases transcription of the proinflammatory cytokine IL-1β in human 157

colon (19) and this cytokine is elevated in patients with C. difficile colitis (20). Ileal administration of 158

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toxin A significantly increased ileal colonic IL-1β protein and mRNA expression in mice (Figure 3A 159

and 3B). Pretreatment of ileal loops with 20 μM, but not 5 or 10 μM of fidaxomicin, significantly 160

reduced C. difficile toxin A induced IL-1β protein levels (Figure 3A) while all concentrations of 161

fidaxomicin (5-20 μM) almost abolished toxin A-induced IL-1β mRNA expression (Figure 3B). 162

Moreover, pretreatment with metronidazole or vancomycin did not affect toxin A-induced IL-1β 163

expression in mouse ileum under the same experimental conditions (Figure 3C). 164

165

OP-1118 reduces C. difficile toxin A mediated tissue damage and IL-1β expression in mouse 166

ileum. 167

To confirm and extend our results on fidaxomicin in toxin A-induced intestinal inflammation, 168

we examined the effect of its primary metabolite OP-1118 on this in vivo toxin A response. OP-1118 at 169

120 μM, significantly reduced C. difficile toxin A mediated ileal damage with reduced histology score 170

(Figure 4A and 4B). Similar to fidaxomicin, OP-1118 at 120 μM, but not 60 μM, significantly reduced 171

C. difficile toxin A-associated IL-1β protein and/or mRNA expression in mouse ileum (Figure 5A and 172

5B). 173

174

Fidaxomicin reduces C. difficile toxin A mediated MAP kinase phosphorylation in mouse ileum. 175

C. difficile toxin A activates MAP kinases, including ERK1/2 in vivo and in vitro (21, 22). 176

Here, we observed induction of ERK phosphorylation in ileal loops exposed to C. difficile toxin A 177

(Figure 6A), while administration of fidaxomicin substantially diminished this response. In contrast, 178

metronidazole and vancomycin did not significantly alter toxin A- mediated ERK1/2 phosphorylation 179

in mouse ileum (Figure 6B). 180

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Fidaxomicin and OP-1118 reduces C. difficile toxin A mediated cell rounding in human colonic 182

fibroblasts. 183

To understand the protective mechanism of fidaxomicin and OP-1118, we examined their 184

ability to affect toxin A-associated cell rounding using human colonic CCD-18Co fibroblasts. 185

Exposure of CCD-18Co fibroblasts to toxin A for 6 hours resulted in cell rounding (Figure 7). Co-186

incubation of cells with fidaxomicin at 20 μM or OP-1118 120 μM partially reduced cell rounding 187

effect of toxin A (Figure 7A). Vancomycin and metronidazole did not prevent toxin A-induced cell 188

rounding (Figure 7B). Similar results were obtained when the fibroblast-like mouse 3T3-L1 189

preadipocytes were used (data not shown). Together, these results indicate that fidaxomicin and OP-190

1118 protect cells against toxin A-mediated cytoskeletal damage. 191

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Discussion: 193

C. difficile mediates CDI and intestinal inflammation by a mechanism involving release of two 194

potent exotoxins, toxin A and toxin B (1, 23). Fidaxomicin is a new antibiotic member of the 195

macrolide family (24), recently approved by the FDA against CDI (25). Although its efficacy is similar 196

to vancomycin, use of fidaxomicin is associated with fewer recurrent episodes of CDI (6). The 197

mechanisms involved in this response are still under investigation, but reduced recurrent CDI rates 198

following fidaxomicin administration may be related to preservation of commensal microflora 199

compared to vancomycin (26, 27), inhibition of sporulation (28) or an inhibitory effect in toxin 200

production by C. difficile (12). 201

202

The in vivo mechanisms by which C. difficile toxins A and B mediate diarrhea and 203

inflammation have been in good part elucidated by studies with relevant experimental models, 204

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including the ileal loop model of toxin A-induced enteritis (29-31). The beneficial effects of 205

fidaxomicin in CDI and the ability of other macrolides to possess anti-inflammatory responses in non-206

gastrointestinal organs (14, 15) led us to hypothesize that fidaxomicin may modulate inflammatory 207

responses against C. difficile toxin A through modulation of signaling pathways regulating mucosal 208

inflammation activated by this toxin. We were unable to test the ability of these drugs to inhibit the 209

effects of toxin B in ileal loops in vivo, since the mouse intestine is insensitive to this toxin in this 210

experimental system (32). Using this model and purified toxin A, we show here that fidaxomicin 211

significantly reduced C. difficile toxin A-mediated histological damage (Figure 1), IL-1β expression 212

(Figure 3), and ERK phosphorylation (Figure 6) in the mouse ileum. We also show that the primary 213

metabolite of fidaxomicin, OP-1118 (33), similar to its parent compound, can also inhibit C. difficile 214

toxin A mediated inflammatory responses in mouse ileum (Figure 4 and 5). Thus, OP-1118 may at 215

least partially mediate the anti-inflammatory effects of fidaxomicin against C. difficile toxin A in the 216

intestine. Our results indicate that pretreatment with metronidazole and vancomycin, commonly used 217

for therapy of CDI, did not significantly alter histologic damage, IL-1β expression or ERK activation 218

in response to toxin A in vivo, and did not affect rounding in response to this toxin in vitro. On the 219

other hand, both metronidazole and vancomycin have been shown to possess anti-inflammatory effects 220

in different in vivo and in vitro conditions (34-37). Different inflammatory stimuli (Staphylococcus 221

areus toxin or LPS) used in the studies above may account for the inability of metronidazole and 222

vancomycin to alter C. difficile toxin A-associated responses shown in our study. 223

Our results demonstrating that fidaxomicin and OP-1118 reduces ERK activation in response to 224

toxin A in vivo, suggest that this pathway may be important to the anti-inflammatory actions of this 225

macrolide during toxin A enteritis (Figure 6). ERK phosphorylation is required to elicit secretion of 226

proinflammatory cytokines in response to C. difficile toxins in vivo and in vitro (21, 22, 38). 227

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Interestingly, another macrolide, azithromycin suppresses IL-1β production and ERK phosphorylation 228

in human peripheral blood mononuclear cells (39), while azithromycin and clarithromycin modulate 229

proinflammatory cytokine secretion in human bronchial epithelial cells, in part through ERK activation 230

(40). Thus, inhibition of proinflammatory cytokine transcription and MAP kinase activation may 231

represent common anti-inflammatory responses of several macrolides (41), including fidaxomicin. 232

This may explain why these two drugs can preserve the normal functions of cells in exposure to C. 233

difficile toxin A. The possibility, however, that the protective effects of fidaxomicin are involved in the 234

lower rates of recurrence of CDI following fidaxomicin treatment remains to be investigated. 235

Our results also indicate that both fidaxomicin and OP-1118, but not vancomycin or 236

metronidazole, reduce the cytopathic effects of C. difficile toxin A in colonic CCD-18Co fibroblasts 237

(Figure 7), suggesting that fidaxomicin and OP-1118 may interfere with the mechanisms involved in 238

this response. The primary molecular mechanism by which C. difficile toxins mediate actin 239

disaggregation and cell rounding following toxin cell surface binding and internalization is 240

glucosylation of Rho, Rac and cdc42 at threonine 37 leading to inactivation of these small GTP 241

binding proteins and eventually to cell rounding (42). Further exploration of specific mechanisms 242

contributing to the inhibitory effect of fidaxomicin and OP-1118 to toxin A-associated cytoskeletal 243

effects is warranted. 244

In summary, fidaxomicin significantly reduces C. difficile toxin A-mediated proinflammatory 245

cytokine expression, ileal tissue damage, and ERK activation in mouse intestinal mucosa. These results 246

strongly suggest that fidaxomicin, like other macrolides, possesses anti-inflammatory activities against 247

C. difficile toxin A independent of its well established antimicrobial effects. 248

249

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Grant Support: 250

This study was supported by Cubist Pharmaceuticals (CP). HWK was supported by a Crohn’s 251

and Colitis Foundation of America Career Development Award (#2691) and NIH grant K01 252

DK084256 grant. SH was supported by a Crohn’s and Colitis Foundation of America student research 253

fellowship (#3831). MC was supported by a Crohn’s and Colitis Foundation of America student 254

research fellowship (#287244). Support was also provided by the Blinder Research Foundation for 255

Crohn’s Disease (CP) and the Eli and Edythe Broad Chair (CP). CPK is supported by NIH Grant RO1 256

AI095256 and XC by a Career Development Award from the Crohn’s and Colitis Foundation of 257

America. 258

259

Acknowledgements: 260

We would like to thank Yuzu Kubota, Deanna Tran and Irene Chang for assisting our 261

experiments. 262

263

Figure Legends: 264

Figure 1 265

Fidaxomicin inhibits C. difficile toxin A-mediated histological damages in ileum. 266

(A) Ileal loops of mice were pre-treated with fidaxomicin (5-20 μM) or 0.8% DMSO followed by toxin 267

A (10 μg per ileal loop) or PBS alone (200 μL). The ileal loops were obtained 4 hours later for H&E 268

staining. Toxin A caused destruction of villous structure that was reduced by fidaxomicin (see arrows). 269

(B) Histology scores were evaluated as mentioned in Materials and Methods section. C. difficile toxin 270

A significantly increased histology score, compared to normal control group. Fidaxomicin at 5-20 μM 271

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dose-dependently reduced histology score in C. difficile toxin A-treated ileal loops. n=6 mice per 272

group. 273

274

Figure 2 275

Metronidazole and vancomycin do not affect C. difficile toxin A-mediated histological damage in 276

mouse ileum. 277

(A) Ileal loops of mice were pre-treated with metronidazole (20 μM), vancomycin (20 μM) or 0.8% 278

DMSO followed by administration of C. difficile toxin A (10 μg per ileal loop) or PBS alone (200 μL). 279

After 4 hours, ileal loops were processed for H&E staining. Toxin A caused destruction of villous 280

structure that was not affected by metronidazole or vancomycin (see arrows). (B) Histology scores 281

were evaluated as mentioned in Materials and Methods. Metronidazole or vancomycin did not alter 282

histological damage score in C. difficile toxin A treated ileal loops. n=6 mice per group. 283

284

Figure 3 285

Fidaxomicin, but not metronidazole and vancomycin, reduces C. difficile toxin A-induced ileal 286

IL-1β expression. 287

(A) C. difficile toxin A (10 μg per ileal loop) significantly induced ileal IL-1β protein expression 288

(p=0.0003) while fidaxomicin (20 μM) significantly reduced C. difficile toxin A induced ileal IL-1β 289

protein expression (p=0.0004). (B) C. difficile toxin A (10 μg per ileal loop) significantly induced ileal 290

IL-1β mRNA expression (p=0.0101) while fidaxomicin significantly reduced C. difficile toxin A 291

induced ileal IL-1β mRNA expression (5 μM p=0.0351; 10 μM p=0.0096; 20 μM, p=0.0384). 292

Fidaxomicin also reduced basal ileal IL-1β protein (p=0.0062) but not mRNA expression. (C) C. 293

difficile toxin A (10 μg per ileal loop) significantly induced ileal IL-1β protein expression (p=0.0036). 294

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Metronidazole and vancomycin (20 μM) did not alter C. difficile toxin A-induced ileal IL-1β protein 295

levels. n=6 mice per group. 296

297

Figure 4 298

OP-1118 has anti-inflammatory effects against C. difficile toxin A in ileum. 299

(A) Ileal loops of mice were pre-treated with OP-1118 (60-120 μM) or 0.8% DMSO followed by C. 300

difficile toxin A (10 μg per ileal loop) or PBS alone (200 μL). Ileal loops were processed after 4 hours 301

for H&E staining. Toxin A caused destruction of villous structure that was reduced by OP-1118 (see 302

arrows). (B) Histology scores were evaluated as stated in Materials and Methods section. C. difficile 303

toxin A significantly increased histology score (p=0.0001), compared to normal control group. OP-304

1118 (120 μM) significantly reduced histology score in toxin A-treated ileal loops (p=0.0004). n=6 305

mice per group. 306

307

Figure 5 308

OP-1118 inhibits C. difficile toxin A induced IL-1β expression in ileum. 309

(A) C. difficile toxin A (10 μg per ileal loop) significantly induced IL-1β protein expression 310

(p=0.0001) in ileal loops that was significantly reduced by OP-1118 (120 μM) (p=0.0286). (B) C. 311

difficile toxin A significantly induced IL-1β mRNA expression (p=0.0101) in ileal loops that was 312

significantly reduced by OP-1118 (120 μM) (p=0.0127). OP-1118 also reduced basal ileal IL-1β 313

protein (p=0.0402) but not mRNA expression. n=6 mice per group. 314

315

Figure 6 316

Fidaxomicin and OP-1118 inhibit C. difficile toxin A-mediated ERK phosphorylation in ileum. 317

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(A and B) Ileal loops of mice were pre-treated with fidaxomicin (20 μM), OP-1118 (120 μM), 318

metronidazole (20 μM), vancomycin (20 μM) or 0.8% DMSO followed by C. difficile toxin A (10 μg 319

per ileal loop) or PBS alone (200 μL). After 4 hours, ileal loops were processed for phospho-ERK 320

immunohistochemistry as described in Materials and Methods section. (A) C. difficile toxin A induced 321

ERK phosphorylation in ileal mucosal tissues that was diminished by fidaxomicin or OP-1118 322

treatment. (B) Metronidazole and vancomycin treatment had no effects on C. difficile toxin A-induced 323

ERK phosphorylation in ileal tissues. n= 6 mice per group. 324

325

Figure 7 326

Fidaxomicin and OP-1118 prevented C. difficile toxin A and B mediated cell rounding. 327

(A) Serum starved CCD-18Co human colonic fibroblasts were treated with DMSO 0.8%, C. difficile 328

toxin A (0.1 ng/ml), fidaxomicin (20 μM) or OP-1118 (120 μM) for 6 hours. The spindle shape of 329

fibroblasts was lost after exposure to toxin A but this change was prevented by co-incubation with 330

fidaxomicin or OP-1118. (B) Serum starved CCD-18Co fibroblasts were treated with DMSO 0.8%, C. 331

difficile toxin A 0.1 ng/ml, metronidazole 20 μM or vancomycin 20 μM for 6 hours. Metronidazole 332

and vancomycin failed to prevent cells from toxin A induced cell rounding. Representative results of 2 333

independent experiments. 334

335

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