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Campylobacter jejuni disrupts protective TLR9 signaling in colonic epithelial cells 1
and increases the severity of DSS-induced colitis in mice. 2
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Jennifer R. O’Hara, Troy D. Feener, Carrie D. Fischer, and Andre G. Buret* 4
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Department of Biological Sciences and Inflammation Research Network, University of 6
Calgary, Calgary, 2500 University Dr., N.W., Calgary, Alberta, Canada T2N 1N4. 7
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Running Title: Campylobacter jejuni and TLR9 signaling. 9
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*Corresponding Author: 11
Dr. Andre G. Buret 12 University of Calgary 13
Bio 336, 2500 University Drive NW 14 Calgary, AB, Canada T2N 4N1 15
Tel: (403) 220-2817 16 Fax: (403) 289-9311 17
E-mail: [email protected] 18 19
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Copyright © 2012, American Society for Microbiology. All Rights Reserved.Infect. Immun. doi:10.1128/IAI.06066-11 IAI Accepts, published online ahead of print on 6 February 2012
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ABSTRACT 28
Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation 29
associated with a dysregulated immune response to commensal bacteria in susceptible 30
individuals. The relapse of IBD may occur following an infection with Campylobacter 31
jejuni. Apical epithelial TLR9 activation by bacterial DNA is reported to maintain 32
colonic homeostasis. We investigated whether a prior C. jejuni infection disrupts 33
epithelial TLR9 signaling and increases the severity of disease in a mild model of DSS 34
colitis in mice. In a further attempt to identify mechanisms, T84 monolayers were treated 35
with C.jejuni followed by a TLR9 agonist. Transepithelial resistance (TER) and dextran 36
flux across confluent monolayers were monitored. Immunohistochemistry, western blot 37
and flow cytometry were used to examine TLR9 expression. Mice colonized by C.jejuni 38
lacked any detectable pathology; however, in response to low levels of DSS, mice 39
previously exposed to C.jejuni exhibited significantly reduced weight gain, increased 40
occult blood and histological damage scores. Infected mice treated with DSS also 41
demonstrated a significant reduction in the anti-inflammatory cytokine, IL-25. In vitro 42
studies indicate that apical application of a TLR9 agonist enhances intestinal epithelial 43
barrier function and this response is lost in C.jejuni-infected monolayers. Furthermore, 44
infected cells secreted significantly more CXCL8 following the basolateral application of 45
a TLR9 agonist. Surface TLR9 expression was reduced in C.jejuni-infected monolayers 46
subsequently exposed to a TLR9 agonist. In conclusion, infection by C. jejuni disrupts 47
TLR9-induced reinforcement of the intestinal epithelial barrier, and colonization by 48
C.jejuni increases the severity of mild DSS colitis. 49
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INTRODUCTION 51
Inflammatory Bowel Disease (IBD), including Crohn’s disease and ulcerative 52
colitis, are chronic, relapsing inflammatory disorders of the gastrointestinal tract (47). 53
Symptoms associated with IBD include diarrhea, bloody stools, abdominal pain, and 54
weight loss. Pathogenesis of this debilitating disease involves the interaction of multiple 55
factors including host immune response, genetics and environmental triggers. A widely 56
held hypothesis is that an inappropriate immune response is generated towards the 57
resident microflora in genetically susceptible individuals (31, 47). Although one specific 58
etiological agent has yet to be identified, several reports suggest that acute bacterial 59
enteritis may initiate or exacerbate disease (1, 9, 12, 13, 30, 46, 49, 51). 60
Campylobacter species, including C.jejuni, are the leading cause of acute bacterial 61
enteritis in the developed world (20). Infection can cause inflammatory diarrhea, fever 62
and abdominal pain (54). While most cases are self-limiting, post-infectious 63
complications including Guillain-Barré syndrome and irritable bowel syndrome can arise 64
(29, 34). Campylobacter species are also commonly isolated from patients with colitis 65
(28, 55) and an acute enteric infection can induce the relapse of IBD (35, 36, 53). 66
Moreover, several recent clinical studies indicate that an acute infection with 67
Campylobacter is a risk factor for the subsequent development of IBD (12, 13, 51). 68
Despite significant advances, the mechanisms by which these events induce the onset or 69
relapse of disease symptoms remain elusive. 70
Bacterial pathogens, like C.jejuni, contain pathogen associated molecular patterns 71
that are recognized by toll like receptors (TLRs). Intestinal epithelial cells are the first 72
line of defense against bacterial pathogens and express several TLRs (25). Activation of 73
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TLRs is typically associated with an inflammatory immune response that is required to 74
clear the invading pathogen. Interestingly, TLRs also recognize the same conserved 75
molecular patterns found in the resident microflora. However, there is a striking contrast 76
in the response elicited by commensal bacteria. Not only does the healthy intestinal 77
epithelium tolerate the presence of the resident microflora, but it also appears to require 78
commensal bacteria recognition via TLRs to maintain intestinal homeostasis (4, 26, 44). 79
Prior studies have demonstrated that bacterial DNA or its synthetic 80
oligodeoxynucleotide analogues (ISS-ODN or CpG ODN) can effectively ameliorate the 81
severity of colitis in various animal models (2, 39, 42). Bacterial DNA and its synthetic 82
analogues are recognized by TLR9. Importantly, mice deficient in TLR9 are more 83
susceptible to colitis (26) and TLR9 genetic polymorphisms are associated with an 84
increased risk of IBD (11, 52), suggesting a critical role for abnormal bacterial DNA 85
sensing in the development of IBD. Whether and how C.jejuni-induced TLR9 disruptions 86
may increase the severity of colitis remains obscure. 87
Given the importance of bacterial DNA sensing by TLR9 in maintaining intestinal 88
homeostasis, we hypothesized that polarized and protective epithelial TLR9 signaling is 89
disrupted following an acute infection with C.jejuni and that this effect may predispose to 90
heightened intestinal inflammation. We first developed an animal model to examine 91
whether an acute infection with C.jejuni increases the severity of a mild experimental 92
model of colitis. We also utilized in vitro cell culture techniques to examine the effects of 93
an infection on TLR9 signaling in intestinal epithelial cells. Our results reveal that an 94
infection with C.jejuni disrupts protective apical TLR9 signaling and sensitizes 95
proinflammatory basolateral TLR9 signaling. Furthermore, colonization by C.jejuni, in 96
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the absence of an inflammatory response, increases the severity of mild DSS-induced 97
colitis in mice. 98
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MATERIALS AND METHODS 100
Reagents, inhibitors and antibodies. Type B CpG oligonucleotide (ODN 2006), 101
a human TLR9 ligand, and ODN 2006 control oligonucleotide, were purchased from 102
Invivogen (San Diego, CA). Bay 11-7085, Apigenin, and LY294002 were purchased 103
from CalBiochem (Gibbstown, NJ), Calbiochem, and Cell Signaling Technology 104
(Danvers, MA), respectively. 105
The following antibodies were used in this study: mouse monoclonal anti- TLR9 106
(IMG-305A, Imgenex, San Diego, CA) for immunocytochemistry and flow cytometry; 107
rabbit polyclonal anti- TLR9 (IMG-431, Imgenex) for immunohistochemistry; rabbit 108
polyclonal anti- TLR9 (GTX111547, GeneTex, Irvine, CA) for western blotting; mouse 109
anti-GAPDH (sc-59540, Santa Cruz Biotechnology, Santa Cruz, CA) for western 110
blotting, Mouse Alexa Fluor 488 and 555- conjugated secondary antibodies (Invitrogen, 111
Carlsbad, CA) were used for immunofluorescence and flow cytometry. HRP-conjugated 112
secondary antibodies (Cell Signaling Technology, Danvers, MA) were used for western 113
blotting. 114
Mice. Wild type C57Bl/6 mice (Charles River, Montreal, QC, Canada), 4-6 115
weeks of age, were housed in a temperature-controlled room. Mice were maintained on a 116
normal 12:12h light-dark cycle and allowed free access to standard lab chow and water. 117
All methods used in this study were approved by the University of Calgary Animal Care 118
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Committee and were carried out in accordance with the guidelines of the Canadian 119
Council on Animal Care. 120
Cell Culture. T84 human colonic epithelial cells (American Type Culture 121
Collection, Manassas, VA) were cultured in DMEM/F-12 (Sigma, St. Louis, MO), 122
supplemented with 10% heat-inactivated fetal bovine serum, 100μg ml-1 streptomycin, 123
100 U ml-1 penicillin, 80μg ml-1 tylosin, and 200mM L-glutamine (all from Sigma). Cells 124
were kept at 37°C and 5% CO2 in 96% humidity. Culture medium was replenished every 125
2-3 days and cells were passaged with 2 x trypsin-EDTA (Sigma). Cells were grown to 126
confluence in 6 well plates for immunoblotting and flow cytometry, or Laboratory-Tek 127
chamber slides (Nalge Nunc International, Naperville, IL) for immunocytochemistry. 128
Cells were also grown on Transwell filter units containing a 1.13-cm2 or 5cm2 semi-129
permeable filter membrane (0.4-μm pore size, Costar, Cambridge, MA) for determination 130
of epithelial permeability, IL-8 secretion or flow cytometry. T84 cells were used at 131
passage numbers 78 to 83. All experiments were performed using serum and antibiotic-132
free media (infection media). 133
Bacteria and infection of mice. Mice were inoculated with 0.1mL of caseamino 134
yeast extract (CYE) broth containing approximately 108 CFU C.jejuni 81-176, plus 2% 135
NaHCO3 by oral gavage on day one and two, as previously described (17). Sham controls 136
were challenged with equal volumes of sterile CYE broth plus 2% NaHCO3. To assess 137
fecal shedding of C.jejuni, fecal pellets were homogenized in PBS, and plated on Karmali 138
agar containing selective supplement SR167 (Oxoid, Nepean, ON). Karmali agar dishes 139
were examined for the presence of C.jejuni growth and scored as positive or negative. 140
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Bacteria and infection of cells. For all experiments, C. jejuni 81-176 was grown 141
from frozen glycerol stocks on Karmali agar plates for 48hrs at 37°C in gas jars (Oxoid) 142
under microaerobic conditions. Inoculum was prepared from the agar plates by 143
suspending Campylobacter in CYE broth for 14-16h (37°C, 100 rpm, microaerobic 144
conditions). Log phase bacteria was centrifuged at 2500x g for 10 min and re-suspended 145
in infection media to achieve a multiplicity of infection of 100. 146
Induction of colitis. Seven days post-infection, colitis was induced by addition 147
of dextran sulfate sodium (DSS, 2% wt/vol; MP Biomedicals; Solon, OH) to the drinking 148
water. The mean DSS-water consumption was monitored for each group. Mice were 149
weighed daily, and monitored for fecal blood and changes in stool consistency. Fecal 150
pellets were collected and examined for the presence of gross blood, and tested for occult 151
blood using a Hemoccult® Sensa kit according to the manufacturer’s instructions 152
(Beckman Coulter, Fullerton, CA). An occult blood score was assigned according to the 153
following criteria: no blood, score = 0; hemoccult positive, score = 2; gross visible blood, 154
score = 3. 155
Tissue Collection and Histology. Mice were euthanised by cervical dislocation 156
12 days following the start of DSS administration. The colon was removed and segments 157
were frozen in liquid nitrogen for ELISA, or fixed in 10% buffered formalin for 158
histology. Paraffin sections (8μm) were cut and stained with haematoxylin and eosin. The 159
severity of colonic inflammation was analyzed in a blinded fashion according to 160
previously described criteria (7), with some modification. Briefly, the severity of 161
inflammation was assigned a histological damage score based on the presence of 162
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ulceration (0-1), inflammatory infiltrate (0-3), edema (0-1), and general tissue 163
architecture, including colonic wall thickening (0-3). 164
Analysis of cytokine levels. IL-17E (IL-25) and IL-17 cytokine levels in colonic 165
homogenates were analyzed by ELISA. Briefly, segments of distal colon from each group 166
were homogenized in ice-cold Tris-HCl buffer containing protease inhibitors (Complete-167
Mini, Roche Diagnostics, Laval QC). Samples were centrifuged at 10,000 g for 30min 168
and the supernatants were collected. Aliquots were stored at -70 until assay. The 169
concentration of IL-25 and IL-17 was analyzed using an enzyme immunoassay according 170
to the manufacturers instructions (Mouse IL-25 kit, Biolegend, San Diego, CA; mouse 171
IL-17A kit, eBioscience, San Diego CA). 172
CXCL-8 ELISA. Control and infected monolayers were treated with apical or 173
basolateral ODN 2006 as described above. After incubation, cell supernatants were 174
collected from the basolateral compartment, snap froze in liquid nitrogen and stored at -175
80ºC. Concentrations of CXCL-8 secreted from polarized T84 cells were analyzed with 176
an enzyme linked immunoabsorbant assay according to the manufacturers instructions 177
(R&D Systems, Minneapolis, MN, USA). 178
In vitro epithelial permeability. Transepithelial resistance (TER) was monitored 179
using an electrovoltohmeter (World Precision Instruments, Sarasota, FL). Confluent 180
monolayers (TER > 1000Ω/cm2) were washed twice with sterile HBSS, followed by the 181
addition of either sterile infection media or infection media containing C.jejuni for 24 hrs. 182
In all cases, C.jejuni was added to the apical compartment. Monolayers were washed 183
twice with sterile HBSS and treated either apically or basolaterally with a TLR9 agonist 184
(5μg/ml, ODN 2006; or control ODN) for an additional 24 hrs. For some experiments, 185
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monolayers were treated apically for one hour prior to administration of the TLR9 186
agonist with the following inhibitors: Bay 11-7085 (10μmol/L; NFκB inhibitor), 187
Apigenin (20μmol/L; MAPK inhibitor), or LY294002 (10μmol/L; PI3K inhibitor). 188
Paracellular permeability was assessed using a non-absorbable fluorescein 189
isothiocyanate (FITC)- conjugated 3kDa dextran probe. Briefly, the apical and basolateral 190
compartments were washed twice with sterile bicarbonate buffered Ringer’s solution. 191
The FITC-dextran probe (500μl, 100μM in Ringer’s solution) was added to the apical 192
compartment and Ringer’s solution (1mL) was added to the basal chamber. After a 3-193
hour incubation (37°C, 5% CO2, 96% humidity), samples were collected from the basal 194
compartment and relative fluorescent units were calculated with a microplate fluorometer 195
(Spectra Max Gemini, Molecular Devices, Sunnyvale, CA). 196
Immunohistochemistry. Segments of colon were fixed in 4% paraformaldehyde 197
overnight at 4°C. Samples were washed, transferred to 20% in PBS overnight at 4°C and 198
embedded in OCT compound (Miles, Elkhardt, IN). Sections of colon (10μm) were cut 199
on a cryostat and mounted onto poly-D-lysine coated slides. The slides were then washed 200
in PBS plus 0.1% Triton X-100 (3 x 10min), blocked in 2% normal goat serum for 1hr, 201
and incubated with rabbit anti-TLR9 (1/100) for 48hrs at 4°C. Sections were washed 202
again and incubated with secondary antiserum for 2hr at room temperature. Slides were 203
examined with a Leica DMR fluorescence microscope. Photos were taken at the same 204
exposure and magnification (40x) using a Retiga 2000x camera (Q Imaging, Surrey, BC). 205
Immunocytochemistry. Cells were washed 3x with sterile PBS, followed by 206
fixation and permeabilisation in Cytofix/Cytoperm (BD Biosciences) for 20 minutes at 207
4ºC. Following three washes in Permwash (BD Biosciences), cells were blocked with 208
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PBS containing 2% BSA for 1hr at room temperature and then stained with mouse anti- 209
TLR9 antibody (1/200) overnight at 4ºC. Cells were washed with Permwash and 210
incubated with secondary antibody (1/1000) for 1 hr at 37 ºC in the dark. Slides were 211
mounted with Aqua PolyMount (Polysciences, Warrington, PA) and visualized using a 212
Leica DMR fluorescence microscope. Micrographs were taken using a Retiga 2000x 213
camera. 214
Western Blot Analysis. T84 cells were washed twice with HBSS and lysed in 215
RIPA buffer (1x PBS, 1% Igepal CA-630, 0.5% sodium deoxycholate, 0,1% SDS; all 216
from Sigma) containing a protease inhibitor tablet (Complete Mini, Roche Diagnostics, 217
Laval, QC) for 30 min at 4°C. Lysates were sonicated and centrifuged at 10,000 g for 10 218
min. Supernatants were collected and protein levels were normalized using a Bradford 219
assay (Bio-Rad, Hercules, CA). Samples were diluted in SDS sample buffer at a 1:1 220
ratio, boiled for 5 min and stored at -20°C. 221
Protein samples were separated by SDS-PAGE (7-10%) and transferred to 222
nitrocellulose membranes (Whatman, Buckinghamshire, England). Membranes were 223
blocked for 1hr in TBS + 0.1% TBS (TBS-T) containing 5% non-fat skim milk, followed 224
by incubation with primary antibodies overnight at 4°C. Membranes were washed with 225
TBS-T and incubated with HRP-conjugated secondary antibodies (1:1000) for 1hr at 226
room temperature. Bands were visualized using the ECL-plus chemiluminescence 227
detection system (GE Healthcare, Pittsburgh, PA) and band density was determined using 228
a Canon CanoScan 4400F scanner and Image J densitometry software. To confirm equal 229
loading, membranes were stripped in 0.5 M acetic acid for 1hr and re-probed for 230
GAPDH. 231
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Flow Cytometry. For evaluation of surface TLR9 expression, T84 cells were 232
detached with 0.2% EDTA in PBS and washed with PBS containing 3% FBS. Live cells 233
were stained with mouse anti-TLR9 antibody (1/100) for 30min at 4°C, washed twice 234
with PBS and incubated with Alexafluor 488-goat anti-mouse secondary antibody 235
(1/1000) for 30min at 4°C in the dark. Expression of TLR9 was measured with a BD LSR 236
II Flow Cytometer. 237
Statistical Analysis. Results are expressed as mean ± SEM and statistical 238
comparisons were conducted with GraphPad Prism Software (GraphPad Software, San 239
Diego, CA). Comparisons between three or more groups were analyzed with a one-way 240
ANOVA followed by a Tukey’s multiple comparison test. Data with a non-parametric 241
distribution were analyzed by a Kruskal-Wallis test followed by a Dunn’s multiple 242
comparison test. Differences of p < 0.05 was considered statistically significant. 243
244
RESULTS 245
An acute infection with C.jejuni increases the severity of DSS-induced colitis 246
in mice. Wild type mice are transiently infected by C.jejuni with no underlying 247
pathology or detectable clinical response (54). In the present study, C.jejuni was detected 248
in the feces of most mice (80%) one week after inoculation. By the end of the study, 249
detection of C.jejuni decreased notably, and bacteria could be detected in 56.7% of 250
infected mice (data not shown). The absence of detectable pathology or clinical response 251
was confirmed in our study, as evidenced by the similar weight gain, disease activity 252
index, and histology scores measured in sham mice, and mice treated with C.jejuni alone 253
(Figure 1 and 2). Mice treated with low levels of DSS had a similar pattern of weight gain 254
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compared to control mice (Figure 1). However, mice previously exposed to C.jejuni 255
demonstrated a significant drop in weight 3 days following the start of DSS treatment. 256
After twelve days of DSS treatment, the infected mice had gained significantly less 257
weight than all other groups (Figure 1). 258
C.jejuni-infected mice treated with DSS also exhibited significantly worse fecal 259
blood scores when compared to mice treated with DSS alone (Figure 1B). Seven days 260
after the start of DSS treatment, 81.3% of infected mice tested positive for occult blood 261
compared with 46.7% of non-infected mice treated with DSS. Furthermore, 262
representative micrographs (Figure 2A) illustrate that non-infected mice treated with low 263
levels of DSS exhibited very mild levels of inflammation (Figure 2B). In contrast, DSS 264
treatment resulted in significant colonic thickening, inflammatory infiltrate and extensive 265
alteration to the tissue architecture in mice exposed to C.jejuni (Figure 2A & B). 266
Effect of a prior C.jejuni infection on cytokine levels in DSS colitis. To 267
determine whether the increased severity of intestinal inflammation was accompanied by 268
altered cytokine production, we measured IL-25 and IL-17 levels in colonic 269
homogenates. Our results show similar levels of colonic IL-25 in sham-, C.jejuni- and 270
DSS-treated mice; whereas there was a significant reduction of IL-25 in C.jejuni- 271
infected mice treated with DSS (Figure 3A). In addition, infected mice treated with DSS 272
exhibited lower levels of IL-17 when compared to sham-treated mice (Figure 3B). 273
TLR9 immunofluorescence is reduced in the colon of C.jejuni-infected mice. 274
To evaluate whether TLR9 expression is disrupted in C.jejuni-infected mice, TLR9 275
protein expression was examined using immunohistochemistry. In sham-treated mice, 276
TLR9 fluorescence was observed throughout the colonic mucosa and was particularly 277
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concentrated in the epithelial cells lining the lumen of the gut (Figure 4). Epithelial TLR9 278
fluorescence was notably reduced in mice exposed to C.jejuni when compared to the 279
sham-treated controls (Figure 4). 280
TLR9 ligand increases intestinal epithelial barrier function in a polarized 281
manner. In vitro experiments were used to assess mechanisms by which a prior 282
infection with C.jejuni increases the sensitivity to gut injury. We examined the effect of 283
TLR9 stimulation on epithelial barrier function in polarized epithelial monolayers. TER 284
and the apical to basolateral flux of a FITC- labeled marker were monitored following 285
administration of ODN 2006 to the apical or basolateral chambers of transwell units. 286
Following the apical administration of ODN 2006, TER was increased by more than 50% 287
(Figure 5A). This was accompanied by a significant reduction in solute flux across the 288
monolayer (Figure 5B), indicating a tightening of the epithelial barrier. When the TLR9 289
agonist was added to the basolateral compartment, no effect on paracellular permeability 290
was observed. The administration of control ODN 2006 had no effect on TER (data not 291
shown). 292
Effect of C. jejuni pretreatment on TLR9- induced increase in epithelial 293
barrier function. To determine whether a prior infection with the C.jejuni could disrupt 294
the TLR9-induced increase in TER, monolayers were pretreated with C.jejuni for 24 295
hours, followed by the administration of ODN 2006, as described above. Monolayers 296
pretreated with C.jejuni failed to exhibit the TLR9-induced increase in TER (Figure 5C). 297
Nor did infected monolayers exhibit a reduction in solute flux in response to apically 298
applied ODN 2006 (Figure 5D). 299
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C.jejuni infection alters the distribution and expression of TLR9 in epithelial 300
cells. TLR9 appears as continuous and uniform staining at the cell periphery in control 301
monolayers and ODN 2006-treated monolayers (Figure 6A). After 24hrs incubation with 302
C.jejuni, there is some redistribution of TLR9 staining to the cytoplasmic compartment. 303
The redistribution of TLR9 is enhanced when the infected monolayers are subsequently 304
treated with ODN 2006. Western Blot analyses of whole cell lysates indicate that infected 305
cells treated with the TLR9 agonist express significantly less total epithelial TLR9 306
(Figure 6B). To further examine the expression pattern of TLR9, live T84 cells were 307
stained and analyzed by flow cytometry. Our results confirm previous studies 308
demonstrating surface expression of TLR9 in control intestinal epithelial cells (Figure 309
7A) and administration of ODN 2006 to C.jejuni-infected cells results in a significant 310
reduction in surface TLR9 fluorescence (Figure 7A, B). 311
Effect of C.jejuni on TLR9- induced CXCL8 secretion. To investigate whether 312
the C.jejuni-induced disruption of TLR9 signaling has an effect on CXCL8, a 313
proinflammatory chemokine upregulated in tissues from IBD patients (37), we examined 314
CXCL8 secretion from T84 cells in response to ODN 2006. We observed increased 315
CXCL8 secretion from control monolayers when a TLR9 agonist was added to the 316
basolateral chamber of the transwell unit. Moreover, significantly more CXCL8 was 317
secreted from infected monolayers in response to basolateral activation of TLR9 when 318
compared to control monolayers (Figure 8). 319
TLR9-Induced Increase in TER is independent of NFκB, MAPK and PI3K 320
signaling pathways. We then investigated possible signaling pathways that are triggered 321
by apical TLR9 to induce the increase in TER. Two common signaling pathways 322
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associated with TLR-induced responses include NF-κB and the mitogen-activated protein 323
kinases (MAPKs) (50). Previous studies have also demonstrated that epithelial TLR2 324
stimulation preserves intestinal epithelial barrier integrity via a PI3K/Akt-dependent 325
pathway (4). However, pretreatment with a NFκB (Figure 2A), MAPK (Figure 9B) or 326
PI3K (Figure 9C) inhibitor did not significantly affect baseline TER, nor did these 327
inhibitors attenuate the TLR9- induced increase in TER. 328
329
DISCUSSION 330
In this study we demonstrate that apical activation of TLR9 increases TER and 331
decreases solute flux across intestinal epithelial cell monolayers. Infection with C.jejuni 332
attenuates the TLR9-induced increase in epithelial barrier function, and this appears to be 333
via the loss of surface TLR9. C.jejuni also sensitizes intestinal epithelial cells to a 334
subsequent pro-inflammatory stimulus, as infected monolayers secrete significantly more 335
IL-8 in response to basolateral activation of TLR9. Furthermore, mice infected with 336
C.jejuni, in which epithelial TLR9 was reduced, were more sensitive to a mild chemical 337
insult, exhibiting significantly worse signs of inflammation. We postulate that these 338
mechanisms may, in part contribute to the C. jejuni- induced exacerbation of symptoms 339
in IBD patients after a bout of acute bacterial gastroenteritis. 340
Campylobacter jejuni is the leading cause of gastroenteritis worldwide and 341
increasing evidence suggests that an acute infection with this common pathogen can 342
initiate the relapse or onset of IBD symptoms (12, 13, 35, 51). Despite its prevalence and 343
link to IBD, relatively little is understood regarding the pathogenesis of C.jejuni 344
infection, and mechanisms by which this pathogen triggers post-infectious complications. 345
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This is, in part, due to the lack of a suitable murine model that reliably reproduces 346
C.jejuni pathogenesis (54). C.jejuni typically colonizes the murine intestine, without 347
inducing an overt inflammatory response (54). The present findings demonstrate an 348
increase in the severity of mild DSS colitis after an acute infection with C.jejuni. 349
Importantly, the low level of DSS used in this study did not induce significant 350
histological damage or weight loss on its own. This suggests that an infection with 351
C.jejuni may not only exacerbate, but also initiate the onset of inflammation in response 352
to an otherwise innocuous stimulus. 353
Increased susceptibility to intestinal inflammation following infection with 354
C.jejuni may be due to a dysregulation in the production of pro- and anti- inflammatory 355
mediators. Members of the Th17 family of cytokines have been linked to the 356
pathogenesis of IBD (10, 21, 45). IL-25 is a member of the IL-17 cytokine family that is 357
reduced in the colonic mucosa of patients with IBD (6). Moreover, treatment with 358
recombinant IL-25 attenuates experimental colitis in mice (6, 32). In the present study, 359
colonic IL-25 levels are significantly lower in DSS-treated mice previously exposed to 360
C.jejuni. Defective bacterial DNA sensing leads to an imbalance in T cell subsets, 361
including a reduction in the IL-17 producing subset of T cells (14). It is possible that 362
C.jejuni infection interferes with commensal bacterial DNA sensing and, by this means, 363
leads to a down-regulation in IL-25 expression. Indeed, we observed a notable reduction 364
in epithelial TLR9 fluorescence in C.jejuni-infected mice. 365
We also observed a reduction in colonic IL-17 levels in the infected mice treated 366
with DSS. Although IL-17 is reported to be upregulated in patients with IBD (10, 45), the 367
exact role of IL-17 in the pathogenesis of IBD remains unclear. For instance, O’Conner 368
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and colleagues demonstrated a protective effect for IL-17A in T cell-driven intestinal 369
inflammation (38), and neutralization of IL-17 is reported to exacerbate DSS-induced 370
colitis in mice (40). IL-17 can also induce the development of intestinal epithelial barrier 371
function in T84 monolayers via an upregulation of claudin-1 and -2 expression (19). 372
More research is needed to determine whether our observation is related to a more acute 373
response that compromises intestinal barrier function, thereby contributing to the 374
increased severity of colitis. 375
Within a healthy host, commensal flora recognition by TLRs is essential for 376
protection against gut injury and maintenance of intestinal homeostasis (44). This 377
phenomena was illustrated in a study demonstrating increased susceptibility to DSS 378
colitis in mice deficient in MyD88, an adaptor molecular crucial for TLR-mediated 379
signaling (44). Previous experiments have demonstrated that TLR9 deficient mice are 380
more susceptible to the development of colitis (26), while administration of CpG ODN 381
significantly ameliorates chemically- induced colonic inflammation in wild type mice (2, 382
39, 42, 43). 383
Significant advances have been made in understanding the pathway by which 384
TLR9 activation limits intestinal inflammation. The polarized expression of TLR9 on the 385
surface of intestinal epithelial cells appears to have an important role in maintaining 386
intestinal homeostasis. TLR9 is expressed on the surface of epithelial cells (8, 26, 48), 387
including both the apical and basolateral membrane of intestinal epithelial cells (26). 388
Interestingly, basolateral, but not apical, activation of TLR9 induces the secretion of 389
CXCL8 (26). We have further characterized the domain specific response of intestinal 390
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epithelial TLR9, and demonstrated that apical activation of this receptor induces an 391
increase in epithelial barrier function, whereas basolateral activation had no effect. 392
An intact intestinal epithelial barrier is required to restrict the translocation of 393
luminal bacteria to the subepithelial compartment. (16). Compromised intestinal 394
epithelial barrier function and the translocation of luminal antigens are key features of 395
IBD (16, 33). Therefore mechanisms that disrupt protective apical TLR9 signaling may 396
destabilize this barrier function and prime the intestine for the development of 397
inflammation. Results from the present study demonstrate that an infection with C.jejuni 398
disrupts TLR9-induced increase in epithelial barrier function. This could then facilitate 399
the translocation of resident bacteria to the sub-epithelial compartment. Indeed, infection 400
with C.jejuni is reported to increase the translocation of noninvasive bacteria through 401
both the paracellular and transcellular pathways (17, 24). 402
The attenuation of apical TLR9-induced increase in epithelial barrier function 403
following an infection with C.jejuni appears to be due to a loss of surface TLR9 404
expression. In the present study, exposure to CpG ODN or C.jejuni alone does not 405
significantly alter TLR9 expression in intestinal epithelial cells. Instead, C.jejuni appears 406
to sensitize intestinal epithelial cells, such that a significant reduction in total and surface 407
TLR9 expression is observed upon the subsequent exposure of the infected cells to a 408
TLR9 agonist. A down-regulation of TLR9 expression and function has also been 409
reported in keritinocytes and cervical cancer-derived epithelial cell lines following viral 410
infection (15). Intriguingly, enteric viral infections are also associated with post-411
infectious onset of symptoms in IBD patients (18, 41). 412
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The strengthening of intestinal epithelial barrier integrity may be a fundamental 413
characteristic of epithelial TLR signaling in response to commensal bacteria (3-5). 414
Activation of TLR2 signaling also exhibits an epithelial barrier protective function, in a 415
PI3K/Akt dependent fashion (4). In contrast to TLR2, the present results indicate that the 416
barrier protective property of TLR9 is independent of the PI3K/Akt pathway. We also 417
examined the role of NFκB and MAPK, which are activated in response to TLR 418
interaction with their respective bacterial ligands. Nevertheless, our results indicate that 419
the TLR9-induced increase in TER does not involve the NFκB or MAPK signaling 420
pathways. 421
It is also known that CpG ODN can elicit an anti-apoptotic effect associated with 422
a TLR9-induced upregulation of heat shock proteins (22, 23). Heat shock proteins have a 423
cytoprotective role in several cell types including intestinal epithelial cells (44) and can 424
protect against intestinal epithelial barrier dysfunction (27). Importantly, mice deficient in 425
MyD88 have significantly reduced levels of heat shock proteins within the colonic 426
epithelium, suggesting the expression of these cytoprotective proteins may be induced 427
directly by TLR signaling in the epithelial cells (44). Whether CpG ODN- TLR9 428
interaction in intestinal cells induces the upregulation of heat shock proteins, thereby 429
regulating the protection of the epithelial barrier integrity, remains to be determined. 430
Our data also suggests that C.jejuni may sensitize the pro-inflammatory 431
basolateral TLR9 pathway, which is associated with secretion of CXCL8 (26). Thus, 432
C.jejuni not only attenuates apical TLR9 signaling, but also appears to enhance the 433
inflammatory response elicited by the basolateral TLR9 pathway. Although we observed 434
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a reduction in surface TLR9, these findings indicate that the loss of TLR9 protein and 435
function may be exclusive to the apical membrane. 436
In summary, our results show that apical epithelial TLR9 plays an active role in 437
protecting epithelial barrier function. This protective response is lost following exposure 438
to C.jejuni. Furthermore, C.jejuni appears to potentiate the proinflammatory CXCL8 439
response elicited by basolateral activation of TLR9. Taken together, these responses 440
indicate that a prior infection with C.jejuni disrupts bacterial DNA sensing via TLR9, 441
which has previously been shown to be essential for tolerance to commensal bacteria and 442
the maintenance of intestinal homeostasis. Importantly, we have developed a murine 443
model that can be used to more closely examine mechanisms by which colonization by 444
C.jejuni contributes to the increased risk of exacerbating or initiating symptoms 445
associated with IBD in susceptible individuals. 446
447
ACKNOWLEDGEMENTS 448
The authors thank Karen Poon for technical assistance with flow cytometry. This 449
work was supported by a grant from the Crohn’s and Colitis Foundation of Canada. J.R.O 450
was the recipient of a CAG/CIHR postdoctoral fellowship. C.D.F. is the recipient of an 451
NSERC graduate scholarship. 452
453
454
455
456
457
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631
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FIGURE LEGENDS 637
638
Figure 1. C.jejuni infection increases clinical disease activity of mild DSS colitis. (A) 639
Percent change in body weight in C57BL/6 mice. Three days after the start of DSS, 640
infected mice exhibited a significant drop in weight when compared to control mice, 641
whereas mice treated with DSS or C.jejuni alone had similar weight gain compared to 642
control mice. By 12 days of DSS treatment, infected mice had gained significantly less 643
weight than all other groups (n = 7-8 mice/group; mean ± SEM; * p<0.05 compared to 644
controls; σ p<0.05 compared to DSS-treated). (B) C.jejuni infected mice have increased 645
incidence of occult blood following DSS treatment when compared to all other groups (n 646
= 15-16 mice/group; mean ± SEM; * p<0.05). 647
648
Figure 2. C.jejuni infection increases the severity of histological inflammation in a model 649
of mild DSS colitis. (A) Representative micrographs of H&E stained colonic sections. 650
Colons from control (upper left panel) and C.jejuni-treated (upper right panel) appeared 651
microscopically similar. Control mice treated with low levels of DSS (lower left panel) 652
displayed mild levels of inflammation. In contrast, infected mice treated with a low level 653
of DSS exhibited extensive inflammation as evidenced by increased mucosal thickening, 654
inflammatory infiltrate and ulceration. Photos are taken at the same magnification. (B) 655
Histological damage scores in control, C.jejuni, DSS and C.jejuni + DSS treated mice. 656
Infected mice treated with DSS demonstrated a significantly increased histopathological 657
score compared to control mice (n=5-8 mice/group; mean ± SEM; * p<0.05). 658
659
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Figure 3. Modulation of Th17 cytokine levels following DSS treatment in C.jejuni-660
infected mice. (A) IL-25 and (B) IL-17 levels were significantly reduced in colonic 661
homogenates from C.jejuni-infected mice treated with DSS (n= 7-8 mice/group; mean ± 662
SEM; *** p< 0.0001, ** p< 0.01; *p<0.05). 663
664
Figure 4. Representative micrographs comparing TLR9 immunfluorescence in the colon 665
of sham and C.jejuni-treated mice. Magnification (40x) and exposure times are the same. 666
TLR9 fluorescence is notably reduced in colonic epithelial cells following infection with 667
C.jejuni (right panel) when compared to sham controls (left panel). 668
669
Figure 5. C.jejuni attenuates TLR9-induced increase in epithelial barrier function. (A) 670
Transepithelial resistance of T84 monolayers was significantly increased in response to 671
the administration of ODN 2006 for 24hrs to the apical compartment of transwell units. 672
Basolateral application of ODN 2006 failed to elicit a change in TER (n=18; mean ± 673
SEM *** p<0.0001). (B) The apical to basolateral flux of a FITC-dextran probe was 674
significantly lower in cells treated with apical ODN 2006 (n=15-16/group; mean ± SEM; 675
*** p <0.0001; ** p<0.001). T84 cells pretreated with C.jejuni for 24hrs did not exhibit a 676
significant change in (C) TER or (D) apical-to-basolateral flux of FITC-Dextran probe 677
following the administration of ODN 2006 for an additional 24hrs to the apical or 678
basolateral chambers (n= 15-18/group; mean ± SEM). 679
680
Figure 6. C.jejuni-infection modulates TLR9 protein expression in epithelial cell 681
monolayers. (A) Representative micrographs of TLR9 immunofluorescence 682
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demonstrating the redistribution of TLR9 away from the cell membrane in C.jejuni-683
infected monolayers (lower left panel). Infected monolayers treated with ODN 2006 684
(lower right panel) exhibit further redistribution, and (B) loss of TLR9 protein levels as 685
determined by immunoblotting. Densitometry data are expressed as % GAPDH 686
(n=9/group; *** p<0.0001; **p<00.1; mean ± SEM). 687
688
Figure 7. Surface TLR9 expression is lost in C.jejuni-infected intestinal epithelial cells 689
following ODN 2006 treatment. Live T84 cells were labeled with an anti-hTLR9 690
antibody and analyzed by flow cytometry for surface detection. (A) Representative FACs 691
histograms comparing surface expression of TLR9 in control (upper left panel), ODN 692
2006 (upper right panel), C.jejuni (lower left panel) and C.jejuni + ODN 2006- treated 693
intestinal epithelial cells (lower right panel). The green histogram represents the level of 694
surface TLR9 expression. The blue filled histogram represents the unlabelled cells. (B) 695
The surface expression of TLR9 is significantly reduced following ODN 2006 treatment 696
in C.jejuni-infected intestinal epithelial cells (n=6/group; mean ± SEM; *p<0.05). 697
698
Figure 8. C.jejuni-infection potentiates ODN 2006-induced CXCL8 secretion. Control 699
and C.jejuni-infected T84 monolayers were treated apically or basolaterally with ODN 700
2006 for 24hrs and CXCL8 secretion was measured by ELISA. Basolateral stimulation 701
with ODN 2006 induces CXCL8 secretion from control cells. Cells previously infected 702
with C.jejuni secrete significantly more CXCL8 in response to basolateral stimulation 703
with ODN 2006 (n=24/group; *p<0.05; mean ± SEM). 704
705
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Figure 9. TLR9-induced increase in TER is independent of NFκB, MAPK and PI3K 706
signaling. Pretreatment of T84 cell monolayers with a (A) NFκB (Bay 11-7085; 707
10μmol/L) (B) MAPK (Apigenin; 20μmol/L) or (C) PI3K (LY294002; 10 μmol/L) 708
inhibitor for 1hr did not attenuate the increase in TER induced by the administration of 709
ODN 2006 to the apical compartment (n=12-16/group; *p<0.05; mean ± SEM). 710
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