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Epithelial PI3K signaling is required for β-catenin activation and host defense 1

against Citrobacter rodentium infection 2

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Short title: PI3K in intestinal epithelial host defense 4

Jeffrey B. Brown1, Paul Cheresh2, Tatiana Goretsky2, Elizabeth Managlia2, Gery R. 5

Grimm2, Hyunji Ryu2, Mojgan Zadeh2, Ramanarao Dirisina2, Terrence A. Barrett2* 6

1Department of Pediatrics, Northwestern University Feinberg School of Medicine 7

/Children’s Memorial Hospital, 2Department of Medicine, Northwestern University 8

Feinberg School of Medicine, Chicago, Illinois, USA 9

Correspondence address: 10

Terrence A. Barrett, MD 11

Department of Internal Medicine, Microbiology and Immunology, and Surgery 12

Northwestern University Feinberg School of Medicine 13

676 N. St. Claire Suite 1400 14

Chicago, IL 60611 15

E-mail: [email protected] 16

Tel: 312-503-0293 17

Fax: 312-503-8423 18

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Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Infect. Immun. doi:10.1128/IAI.01025-10 IAI Accepts, published online ahead of print on 22 February 2011

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ABSTRACT 24

Citrobacter rodentium (C. rodentium) infection of mice induces cell mediated immune 25

responses associated with crypt hyperplasia and epithelial β-catenin signaling. Recent 26

data suggest that phosphatidylinositol-3-kinase (PI3K)/Akt signaling cooperates with 27

Wnt to activate β-catenin in intestinal stem and progenitor cells through phosphorylation 28

at Ser552 (P-β-catenin552). Our aim was to determine whether epithelial PI3K/Akt-29

activation is required for β-catenin signaling and host defense against C. rodentium. 30

C57/BL6 mice were infected with C. rodentium and treated with DMSO (vehicle control) 31

or with the PI3K inhibitors, LY294002 or wortmannin. Effects of infection on PI3K 32

activation and β-catenin signaling were analyzed by immunohistochemistry. Effects of 33

PI3K inhibition on host defense were analyzed by quantification of splenic and colon 34

bacterial clearance, and adaptive immune responses were measured by RT-PCR and 35

ELISA. Increased numbers of P-β-catenin552-stained epithelial cells were found 36

throughout expanded crypts in C. rodentium colitis. We show that inhibition of PI3K 37

signaling attenuates epithelial Akt activation, Ser552 phosphorylation and activation of 38

β-catenin, and epithelial proliferative responses during C. rodentium infection. PI3K 39

inhibition impairs bacterial clearance despite having no impact on mucosal cytokine 40

(IFN-γ, TNF, IL-17, IL-1β) or chemokine (CXCL1, 5, 9, 10) induction. The results 41

suggest that host defense against C. rodentium requires epithelial PI3K activation to 42

induce Akt-mediated β-catenin signaling and clearance of C. rodentium, independent of 43

adaptive immune responses. 44

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Phosphatidylinositol-3-kinase (PI3K)/Akt signaling induces cell proliferative, growth, and 47

survival signals in intestinal epithelial cells (IEC)(3, 17, 61). PI3K phosphorylates PIP2 48

to generate PIP3, which functions as a ligand to recruit pleckstrin homology (PH)-49

domain-containing proteins to the inner surface of the cellular membrane. Chief among 50

these is Akt, a serine/threonine kinase that is itself phosphorylated upon activation. Akt 51

has emerged as a critical signaling node in mammalian cells and mediates the majority 52

of PI3K’s downstream effects. Amongst its many pro-proliferative functions, Akt 53

signaling enhances mRNA stability and protein synthesis (via mTOR and S6 kinase) 54

and uptake of energy substrates(36). Activated Akt opposes apoptotic signaling by 55

phosphorylating and sequestering bcl-xL/bcl-2-associated death promoter homolog 56

(BAD) and Forkhead box factors (FOXO) in the cytoplasm. Recent findings suggest 57

activated Akt also acts in the nucleus by phosphorylating substrates, such as β-catenin, 58

that increase transcription of target genes associated with proliferation (e.g. cyclinD1, c-59

Myc), tissue remodeling (e.g. MMP7), and host defense (e.g. cryptdins, Muc2, intestinal 60

trefoil factor)(15, 18, 33). 61

Wnt/β-catenin signaling is critical to the maintenance of intestinal crypt cell 62

proliferation. Wnt ligands bind intestinal stem cells (ISC) and crypt epithelial progenitor 63

cells (PC) and prevent GSK3β-dependent N-terminal phosphorylation and proteosomal 64

degradation of β-catenin(22, 29, 55, 60). When degradation is impaired by Wnt 65

signaling, β-catenin translocates to the nucleus (a hallmark of activated canonical 66

Wnt/β-catenin signaling(43)) where it initiates transcription of Wnt target genes such as 67

c-Myc and cyclinD1(23, 29, 47). Upon division, ISCs and PCs give rise to rapidly cycling 68

transit-amplifying cells(4, 6, 37). Previous studies showed that deletion of the 69


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Phosphatase and Tensin homolog (PTEN), a negative regulator of PI3K, increases 70

PI3K/Akt activation and enhances nuclear β-catenin accumulation(42). More recently, 71

He et al reported that PTEN deficiency increases PI3K/Akt activation resulting in 72

excessive proliferation and crypt fissioning within small bowel (SB) polyps(24). To 73

investigate the interface between activated PI3K/Akt and β-catenin stabilization, 74

researchers developed a phospho-specific antibody that identifies β-catenin 75

phosphorylated by Akt at Ser552 (P-β-catenin552)(24). Compared to N-terminal 76

phosphorylation which targets β-catenin for degradation, C-terminal phosphorylation at 77

Ser552 is detected on nuclear β-catenin associated with enhanced transcription of Wnt/ 78

β-catenin target genes(18, 24). Staining of tissue from SB polyps found P-β-catenin552 79

within nuclei of dividing (activated) ISCs and PCs in crypt bases and at points of crypt 80

branching(24). Taken together, these results suggest PI3K/Akt activation cooperates 81

with Wnt to promote β-catenin signaling in ISCs and PCs. 82

Overactive PI3K signaling occurs in a variety of tumor types, including breast, 83

lung, ovarian, pancreatic, prostate, and colon cancers(1, 14, 20, 41, 46, 57, 62). 84

Furthermore, defects in PTEN occur in a wide spectrum of human cancers(10). Recent 85

studies by Mayer and colleagues suggest PI3K/Akt signaling is upregulated in crypt IEC 86

in chronic ulcerative colitis and active Crohn’s disease(11). We recently reported that 5-87

aminosalicylic acid treatment impairs Akt-mediated β-catenin signaling and prevents the 88

development of colitis-associated dysplasia(8). These data suggest that activation of 89

PI3K is an important step in activation of IEC β-catenin signaling in dysplasia and 90

cancer. As activating mutations of the p110 subunit of PI3K are amongst the most 91

common detected in all of human cancer(27, 45, 46), several newly developed cancer 92


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therapeutics target this pathway(30, 35). Therefore, molecules that interfere with PI3K 93

signaling may be chemopreventive in inflammation-induced cancer by inhibiting β-94

catenin signaling and impairing ISC and PC activation. 95

Despite literature examining PI3K and β-catenin in crypt maintenance, cell 96

survival and malignancy, little is known about its role in intestinal inflammation. This is 97

especially important in infectious enterocolitis where maintenance of the epithelial 98

barrier is critical to survival. Citrobacter rodentium (C. rodentium) is classified as an 99

attaching and effacing (A/E) pathogen whose colonization of colon epithelia results in 100

epithelial hyperplasia and mucosal inflammation(7). C. rodentium induces crypt 101

hyperplasia, promotes PI3K recruitment to lipid rafts(51) and increases levels of nuclear 102

β-catenin in crypt IEC(49, 50). Studies here address the possibility that efficient host 103

defense responses to C. rodentium infection involve PI3K-induced β-catenin activation. 104

Our results indicate C. rodentium infection increases IEC levels of activated P-Akt and 105

nuclear P-β-catenin552. To determine the requirement for PI3K regulation of epithelial 106

β-catenin signaling in host defense, we inhibited PI3K signaling in mice inoculated with 107

C. rodentium. Loss of PI3K signaling in infected mice reduced IEC Akt and β-catenin 108

activation and attenuated IEC BrdU incorporation. Inhibition of PI3K signaling delayed 109

bacterial clearance while not affecting inflammatory responses. Taken together, these 110

results suggest PI3K-mediated Akt and β-catenin signaling play critical roles in 111

epithelial-specific host responses to enteric infection. 112

Materials and Methods 113

Animals 114


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6-8 week old C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME) were 115

maintained within infection-controlled containment housing in the Northwestern 116

University animal care facility. All experiments were approved by the Animal Care and 117

Usage Committee of Northwestern University. 118

Induction of Colitis and Study Design 119

C. rodentium strain DBS120(48), a kanamycin-resistant strain (a gift from David 120

Schauer, Boston, MA), were prepared by shaking at 37ºC overnight in LB broth. 121

Bacterial concentration was determined by measuring absorbance at optical density 122

600. Colony forming units (CFU’s) were confirmed through serial dilution and plating. 123

Mice were fasted for 8 hours prior to inoculation with 109 CFU’s in 200 uL sterile PBS. 124

During the study, mice were allowed free access to food and drinking water. Mice were 125

divided into 2 groups: Group 1 received 200 µL intraperitoneal (i.p.) injections of 126

dimethyl sulfoxide (DMSO) diluted 1:4 in phosphate buffered saline (PBS) (vehicle 127

control) on days 5, 8, and 13 after infection, and Group 2 received 1 mg (appr. 50 mg/kg 128

body weight) LY294002 (Sigma, St. Louis, MO) dissolved in DMSO diluted 1:4 in PBS. 129

In separate studies, a parallel set of mice in Group 2 received 0.1 mg/kg wortmannin 130

(Sigma, St. Louis, MO) dissolved in DMSO diluted 1:5 in PBS on days 4, 6, 8, and 10. 131

In studies to specifically examine IL-22 production, injections of DMSO or LY294002 132

were given on days 1, 3, and 5 after infection and mice sacrificed for analysis on days 4 133

and 7. 134

Histologic and Immunohistochemical (IHC) Analysis 135


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A distal 1 cm piece of colon was removed from each mouse, fixed in 10% neutral 136

buffered formalin, routinely processed, sectioned at 5 µm, and stained with H&E for 137

microscopic examination. Crypt lengths were measured by micrometric sampling of at 138

least 15 crypts per mouse. Histologic colitis scores were determined by measuring the 139

severity of epithelial ulceration (0, none; 1, epithelial desquamation; 2, erosions with 140

gaps of 1-10 IECs; 3, ulcers >10 IECs long), the extent of inflammatory cell infiltration 141

(0, normal; 1, mild infiltration of the mucosa; 2, moderate-severe infiltration of the 142

mucosa; 3, mild infiltration of the submucosa; 4, moderate-severe infiltration of the 143

submucosa; 5, transmural infiltration into the serosa), and extent of submucosal edema 144

(0, none; 1, <200 µm; 2, 200-450 µm; 3, >450 µm). BrdU, P-Akt, and P-β-catenin552 145

IHC staining was performed as previously described(32). 146

Immunofluorescent Analysis 147

Paraffin tissue sections were subjected to antigen retrieval as described for P-β-148

catenin552 previously. Slides were incubated in rabbit anti-Myc Ab (Millipore/ Upstate, 149

Temecula, CA) diluted 1:700 in PBS for 48 hours at 4˚C. After washes with PBS, HRP-150

conjugated polymeric secondary antibody (EnVision anti-Rabbit HRP, DAKO) was 151

applied for 60 minutes at room temperature. Slides were fluorescently labeled with 152

Alexa 488 via Tyramide signal amplification, according to the manufacturer’s 153

instructions (Invitrogen), washed with PBS, stained with Hoechst 33342 (Invitrogen), 154

washed in distilled water and mounted in aqueous medium. Slides were imaged with a 155

Nikon C1 confocal microscope. 156

Colony-forming Unit Counts 157


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At selected time points, colons were removed from anus to cecum and tissue and 158

stool were homogenized using a Polytron tissue homogenizer (Kinematica, 159

Switzerland). Spleens were homogenized through a 100µm mesh filter. Homogenates 160

were serially diluted, plated in duplicate on MacConkey agar (Fluka, Steinheim, 161

Germany) containing 40 µg/mL kanamycin (Fluka) (C. rodentium strain DBS120 carries 162

a kanamycin resistant plasmid), and colonies counted after 18 hours of incubation at 163

37ºC. 164

Real Time PCR Analysis 165

Tissue RNA was isolated using Trizol reagent (Invitrogen). Real-time (RT) PCR 166

analysis was performed using a qScript RT kit (Quanta, Gaithersburg, MD), QuantiTect 167

SYBR green PCR kit (Quigen), and primers on an ABI 7500 Real-time PCR System. 168

Primers were designed to span genomic DNA intron junctions for specific amplification 169

of mRNA. Samples were normalized using Gapdh. Fold increases were calculated 170

using the ddCT method. Primer sequences available upon request. 171

Colon explant culture ELISA 172

1 cm pieces of distal colonic tissue were weighed, irrigated with PBS, shaken at 173

room temperature in RPMI containing 50 µg/mL gentamicin for 30 minutes, and 174

distributed (0.05 g per well) into 24-well plates in 1 mL of RPMI supplemented with 5% 175

fetal bovine serum, 50 µg/mL gentamicin, and 1% penicillin/streptomycin/amphotericin B 176

(GIBCO, Grand Island, NY) for 20 hours at 37°C. Supernatants were collected and 177

stored at −80°C until protein quantification via ELISA kits (R&D, Minneapolis, MN). 178

Statistical Analysis 179


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Data were analyzed with GraphPad Prism software by means of ANOVA 180

followed by Tukey’s range test when more than two groups were compared; otherwise, 181

the Student’s t test was used. P-values ≤ 0.05 were considered statistically significant. 182

Unless specified, values represent mean ± standard error of the mean (SEM). Unless 183

specified, data are representative of at least 2 independent experiments. 184

Results 185

C. rodentium colitis induces PI3K and β-catenin signaling in hyperplastic crypts 186

C. rodentium induces pronounced colonic crypt hyperplasia in mice. We 187

postulated that C. rodentium elicits epithelial proliferation by activating PI3K and β-188

catenin signaling. To examine this possibility, tissues were stained for BrdU 189

incorporation, P-Akt and P-β-catenin552 (Fig. 1A). Results indicate that crypt lengths 190

nearly doubled within one week of infection (baseline to day 7, mean ± SEM: 92 ± 10 to 191

176 ± 7 µm, Fig. 1B), then peaked within weeks two and three (239 ± 14 µm). 192

Coincident with crypt lengthening, numbers of cells staining for BrdU, P-Akt and P-β-193

catenin552 increased 3-5 fold within the first week, peaked by day 14 and declined 194

thereafter (Fig. 1C-E). Examination of timed sections in the first two weeks suggested 195

C. rodentium induced IEC proliferation within mid and upper crypt regions. By 196

comparison, BrdU-stained cells receded from upper crypts one week later (day 21). 197

Likewise, P-Akt and P-β-catenin552 staining reached apical crypt regions by day 14 198

before diminishing. Examination of alcian blue staining indicated that numbers of goblet 199

cells decreased during the first two weeks of infection (Fig. 1F) as previously 200

described(5, 34). To test whether the increase in BrdU and PI3K/β-catenin signaling 201


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was simply an effect of increased crypt length, numbers of positive cells were calculated 202

based on average crypt lengths. Increased crypt lengths in inflamed mice reduced the 203

absolute differences in counts but data remained consistent with total crypt counts (data 204

not shown). These results raised the possibility that IEC proliferative responses to C. 205

rodentium were induced by PI3K and β-catenin signaling in mid and upper crypts. 206

PI3K signaling is required for P-β-catenin552 expression and crypt proliferation in 207

C. rodentium-induced colitis 208

To examine the role of PI3K signaling in epithelial responses to C. rodentium 209

infection, infected mice were treated with vehicle (DMSO) or the selective PI3K inhibitor, 210

LY294002, during the first two weeks of infection (Fig. 2A). A third group of uninfected 211

mice were treated with an identical dosing schedule of LY294002 over two weeks to 212

assess the effect of PI3K inhibition on normal IEC turnover. Morphometric analysis data 213

(Fig. 2B) indicate that crypt lengths were unaffected by PI3K blockade in uninfected 214

mice. In contrast, PI3K inhibition reduced crypt elongation by 36% in infected mice. IHC 215

results show that PI3K inhibition had no effect on the distribution of staining in 216

uninfected mice, but attenuated the vertical expansion of BrdU, P-Akt and P-β-217

catenin552 staining into upper crypts of C. rodentium infected mice. Cell counting of 218

BrdU, P-Akt and P-β-catenin552 stained sections demonstrated PI3K inhibition reduced 219

numbers of BrdU+ cells by 67% (Fig. 2C) and active Akt and β-catenin signaling by 50% 220

and 42% respectively (Fig. 2D,E). Additionally, we observed that reductions in goblet 221

cell numbers in C. rodentium–infected mice were partially reversed as alcian blue 222

stained cells reappeared in the upper crypt of LY294002-treated mice (Fig. 2F). To 223

confirm that changes in PI3K signaling measured per crypt were solely due to impaired 224


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proliferation (i.e. fewer numbers of IEC), BrdU, P-Akt, and P-β-catenin552 positive cells 225

were enumerated on a per IEC basis. Results were similar to crypt counts revealing 226

PI3K inhibition reduced the absolute number of proliferating epithelial cells with β-227

catenin signaling (data not shown). Finally, to test whether the PI3K-inhibition affected 228

levels of IEC apoptosis, sections were TUNEL stained. Data showed no difference in 229

apoptotic indices after LY294002 treatment (Figure 3). Taken together, these data 230

suggest that PI3K activation induced IEC proliferation via Akt-dependent 231

phosphorylation of β-catenin in C. rodentium colitis altering IEC cell death. The net 232

result was to increase crypt lengths. 233

Functional activity of β-catenin is dependent on PI3K-mediated Ser552 234

phosphorylation 235

Our data in both human inflammatory bowel disease and experimental IL-10-/- 236

murine colitis(8, 32) as well as data from Fang et al(18) suggests that Ser552 237

phosphorylation of β-catenin stimulates transcriptional expression of Wnt/β-catenin 238

target genes. c-Myc and cyclinD1 are Wnt/β-catenin target genes involved in cell 239

proliferation(23, 59). In recent studies, Clevers and colleagues reported that c-Myc 240

protein expression denotes proliferative transit amplifying cells in the mid-crypt(39). To 241

assess the functional consequences of PI3K inhibition on epithelial c-Myc and cyclinD1 242

expression, colon sections were stained for c-Myc and cyclinD1 (Fig. 4). In uninfected 243

mice, c-Myc and cyclinD1 stainings localized to IECs immediately above (c-Myc) or 244

within (cyclinD1) the crypt base. In response to infection, both c-Myc and cyclinD1 245

levels increased throughout mid and upper crypts. Data also reveal that LY294002 246

treatment inhibited the expression of both proteins in mid and upper crypts. These 247


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findings suggest C. rodentium infection induced expression of c-Myc and cyclinD1 in 248

transit amplifying cells through a PI3K-dependent process. Together, data suggest that 249

C. rodentium-induced IEC PI3K activation stimulated epithelial proliferation by 250

stimulating Akt and β-catenin signaling. 251

PI3K signaling is required for host defense against C. rodentium 252

To test the role of PI3K signaling in host responses to C. rodentium, body 253

weights and C. rodentium bacterial loads in control and LY294002-treated mice were 254

measured up to 28 days after infection. Infection caused transient weight loss at day 4 255

(average body weight decreased by 0.5%) followed by weight gain (Fig. 5). Infected 256

mice treated with LY294002 failed to gain weight at day 7, followed by further weight 257

loss that continued until day 16 whereupon a gradual recovery ensued. To determine 258

whether toxicity from LY294002 itself caused weight loss, uninfected WT mice were 259

treated with 3 doses of LY294002 and weights were compared to control infected mice. 260

LY294002-treated uninfected mice gained weight in parallel with untreated mice. We 261

then considered the possibility that weight loss in LY294002-treated infected mice 262

resulted from prolonged bacterial infection. Consistent with this interpretation, we found 263

that DMSO- and LY294002-treated mice had comparable colonic and splenic bacterial 264

burdens at day 7, but LY294002 treatment delayed clearance of colonic bacteria at both 265

day 14 (4-fold more CFU’s) and day 18 (300-fold more CFU’s) (Fig. 6A), which 266

correlated with the weight divergence (day 10) between the two groups. Histologic 267

findings in LY294002-treated mice suggested that attenuated PI3K signaling impaired 268

epithelial restitution (Fig. 2) resulting in more severe, prolonged ulceration and impaired 269

barrier function (Fig. 7). We then tested whether compromised mucosa correlated with 270


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enhanced bacterial translocation. Analysis of splenic C. rodentium levels revealed that 271

infected control mice cleared splenic bacteria by day 18. By comparison, splenic 272

bacterial burdens in LY294002-treated mice remained elevated at days 14 and 18 with 273

clearance at day 21 (Fig. 6B). Together, these data support the hypothesis that 274

epithelial PI3K signaling helps to prevent C. rodentium bacterial invasion. 275

Host adaptive immune responses are not affected by treatment with LY294002 276

Previous studies indicate that cytokine(13, 21, 31, 52, 53, 56, 58) and 277

chemokine(54)-mediated adaptive immune responses promote host defense against C. 278

rodentium. To determine the effect of PI3K inhibition on adaptive mucosal host defense 279

to C. rodentium, mucosal Th1, Th17, and macrophage cytokine levels, as well as 280

chemokine responses were assessed in infected mice. Data in Figure 8A show that 281

IFN-γ, TNF, IL-17, and CXCL9 mRNA levels peaked within fourteen days of C. 282

rodentium infection. Furthermore, treatment with LY294002 during this period did not 283

affect induction of these inflammatory mediators. In fact, cytokine and chemokine 284

mRNA levels for all genes assessed increased and/or remained elevated in LY294002-285

treated mice, including CXCL1, CXCL5, CXCL10, IL1-β, and iNOS (data not shown). 286

Protein quantification by colon explant culture ELISA (Fig. 8B) at day 14 revealed that 287

C. rodentium infection also increased cytokine and chemokine protein levels 288

independent of PI3K. Interestingly, mucosal IFN-γ, TNF, CXCL9 and 10, and iNOS 289

mRNA levels increased after LY294002 treatment was stopped. Examination of tissues 290

during this period revealed that tissue remained inflamed (histologic colitis score 5.8 ± 291

0.2 vs. 6.3 ± 0.3, C. rodentium-infected vs. C. rodentium-infected with LY294002 292

treatment, p=NS, (Fig. 7B). One possibility suggested by assessments of bacterial 293


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counts (Fig. 6) was that mucosal inflammation remained elevated to allow host 294

defenses to recover from PI3K inhibition. To test whether the increased cytokine and 295

chemokine levels seen in LY294002-treated infected mice was due to LY294002, colon 296

mRNA from uninfected mice that received 3 doses of LY294002 was compared to 297

control uninfected mice. Data showed no increase in cytokine/chemokine mRNA levels 298

after LY294002 treatment (data not shown). 299

Recently, IL-22 has been implicated as a major determinant during the first week 300

of mounting host defenses to C. rodentium(63). We considered this mechanism of host 301

defense to be relevant because the IL-22 receptor is expressed on epithelial cells(2). 302

Data in Fig. 9 show that at both day 4 and 7 after infection, IL-22 induction was 303

unaffected. 304

Finally, we considered that PI3K inhibition may be affecting innate immune 305

signaling, including antimicrobial function of paneth cells and enterocytes. Data in Fig. 306

8C show that both Reg3γ (Paneth cells) and MMP-7 (enterocytes) induction was 307

unaffected. In fact, early enhanced induction of Reg3γ mRNA (day 7) and prolonged 308

induction of MMP-7 were noted. These data support the notion that PI3K signaling is 309

critical to epithelial host defenses independent of adaptive inflammatory responses. 310

Alternative PI3K inhibition with wortmannin corroborates impaired bacterial 311

clearance with LY294002 312

Although we have demonstrated that PI3K inhibition by LY294002 treatment 313

impairs mucosal host defense by inhibiting epithelial proliferative responses while 314

sparing host inflammatory responses, it remained possible that LY294002 was acting 315


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through a mechanism independent of the epithelial compartment or independent of 316

PI3K. Therefore, we performed a similar set of experiments interrupting PI3K signaling 317

with the irreversible PI3K inhibitor, wortmannin. Morphometric analysis data (Fig. 10A) 318

indicate that crypt lengths were unaffected by PI3K blockade with wortmannin in 319

uninfected mice. In contrast, PI3K inhibition reduced crypt elongation by 43% in infected 320

mice, consistent with the impaired epithelial proliferative responses seen in LY294002-321

treated mice. Furthermore, wortmannin treatment resulted in similar wasting state to 322

LY294002, while having no effect on uninfected mice (Fig. 10B). As expected, colonic 323

bacterial clearance was delayed at day 18, and systemic levels of bacteria were greater 324

at day 14 and remained present ay day 18 (Fig. 10C). To measure whether wortmannin 325

spared host adaptive immune responses in a manner similar PI3K inhibition with 326

LY294002, colonic mRNA levels of IFNγ, CXCL9, and iNOS were measured at day 14 327

and 18. Data in Fig. 10D show that PI3K inhibition did not affect induction of these pro-328

inflammatory genes. This data corroborates findings in LY294002-treated mice and 329

suggests PI3K-mediated signaling plays a critical role in epithelial-specific host 330

responses to enteric infection. 331

DISCUSSION 332

The present study proposes a novel view of the role of PI3K signaling in 333

mediating epithelial responses to C. rodentium. Previous studies suggested that PI3K 334

signaling induces epithelial proliferation in response to growth factor, TNF and toll-like 335

receptor 4 ligation(16, 19, 25, 36). Data from epithelial co-culture studies indicate 336

mucosal T cells from Crohn’s disease tissue stimulate IEC PI3K signaling(11, 12). 337

Results presented here show that IEC PI3K signaling was induced during the period of 338


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C. rodentium-induced inflammation associated with crypt elongation and enhanced IEC 339

proliferation (Fig. 1, 2). Reductions in crypt elongation and IEC proliferation in 340

LY294002-treated mice indicated that PI3K signaling was required for the proliferative 341

responses induced by C. rodentium. Proliferative responses may be pathogen driven to 342

facilitate increased surface area for C. rodentium colonization. However, we found that 343

blocking PI3K signaling/proliferation increased pathogen burden and prolonged mucosal 344

inflammation (Fig.’s 6-8). Thus, our findings are consistent with the notion that C. 345

rodentium-stimulated IEC PI3K signaling induces proliferation as a component of 346

mucosal host defenses needed to eliminate infection and resolve mucosal inflammation. 347

Data from our group(8, 24) and others(18) suggest that PI3K signaling serves to 348

enhance Wnt-mediated β-catenin activation. Studies in PTEN-deficient mice suggested 349

that enhanced PI3K signaling in IEC led to SB polyp formation. The detection of IEC 350

with nuclear P-β-catenin552 at points of crypt fissioning suggested that Akt-mediated 351

phosphorylation of β-catenin enhanced nuclear stabilization and activation. The anti-P-352

β-catenin552 Ab employed in these studies was used here to visualize cells with C-353

terminal phosphorylation of nuclear β-catenin in crypts of mice with C. rodentium 354

infection. Sellin et al previously reported enhanced levels of P-β-catenin552 in C. 355

rodentium colitis(49, 50). Studies here suggested that the induction of P-β-catenin552 in 356

crypts was regulated by PI3K signaling. In addition, the data were consistent with the 357

notion that PI3K mediated β-catenin activation played a key role in mounting robust 358

crypt epithelial proliferative responses during C. rodentium infection. The localization of 359

cells with nuclear P-β-catenin552 in the upper crypts of infected mice suggests that PI3K 360

signaling induced β-catenin activation in PCs within transit amplifying populations. We 361


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postulate that PI3K signaling in upper crypt IEC was induced by inflammatory signals 362

received from the local environment. Of the many downstream functions of PI3K/Akt 363

signaling (protein stabilization, reduced apoptosis, etc), we suspect that Akt interacts in 364

a multi-component complex with β-catenin in the nucleus to enhance activation. In this 365

model, we suspect Akt activation did not promote nuclear localization of β-catenin, 366

which occurs during Wnt signaling. Rather, our results suggest that β-catenin 367

phosphorylation by Akt in the nucleus initiates transcription of pro-proliferative genes 368

(e.g. c-Myc, cyclinD1). This signal cooperates with effects of Akt in the cytosol (e.g. 369

mTOR activation) that promote cell cycle progression and replication. Together, the 370

data suggest that inflammatory signals derived from the surrounding milieu promote β-371

catenin signaling through a PI3K-dependent pathway. 372

The data presented show that PI3K inhibition was associated with increased 373

systemic levels of C. rodentium. Because it is not possible to confirm unperturbed 374

function of all adaptive pro-inflammatory pathways(13, 21, 52-54, 56, 58) and innate 375

host defense factors (e.g. cathelicidin, TFF)(15, 26) or unknown effects of LY294002 376

and wortmannin, it remains possible that systemic PI3K inhibition may have contributed 377

to increased systemic bacterial load. However, preservation of Th1, Th17, 378

macrophage, paneth cell, epithelial, and chemokine mRNA levels suggested that PI3K 379

inhibition did not impair mucosal inflammatory responses. Rather, we postulate that 380

PI3K inhibition reduced epithelial proliferative responses and barrier function. This 381

hypothesis is consistent with results from Podolsky and colleagues who found that 382

inhibition of IEC PI3K/Akt signaling caused disruption of tight junctions following toll-like 383

receptor 2 stimulation(9). Similarly, Resta-Lenert et al found that PI3K inhibition 384


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reversed the protective effect of probiotics on barrier function in epithelial 385

monolayers(44). The findings presented here add to the potential mechanisms for the 386

role of PI3K signaling in epithelial barrier integrity. The data suggest that PI3K signaling 387

may enhance mucosal barrier function in infectious colitis by promoting epithelial 388

restitution. 389

A/E-type enteric pathogens such as Enteropathogenic and Enterohemorrhagic E. 390

coli generate substantial global morbidity (e.g. infantile diarrhea, hemolytic uremic 391

syndrome) and mortality, resulting in as many as 1 million deaths per year world 392

wide(28, 38, 40). Elucidating mechanisms required for host defense against A/E 393

pathogens may impact global health. Data here utilize C. rodentium, an A/E murine 394

pathogen, to show that PI3K-dependent activation of β-catenin and subsequent 395

epithelial proliferative responses are required for normal host-defense in C. rodentium 396

colitis. In the current study, it remains unclear whether or not the eventual clearance of 397

bacteria was due to restitution of PI3K signaling after cessation of LY294002 treatment, 398

or if enhanced inflammatory responses were enough to compensate for impaired 399

epithelial signaling. Regardless, given the global morbidity and mortality associated 400

with A/E enteric pathogens and the fact PI3K signaling may be targeted in future 401

chemotherapeutic agents(35), it is crucial to better understand its role in non-malignant, 402

inflammatory processes. 403

Acknowledgments 404

This work was supported by National Institutes of Health Grants DK54778, DKAI061701 405

(to T.A.B.) and DK066161 (to J.B.B.). 406


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Figure 1. C. rodentium-induces epithelial hyperplasia and PI3K signaling. Colon tissue from control (Day 0) or mice inoculated with C. rodentium are shown. (A) From left to right are representative photomicrographs of tissue from infected mice at specified time points stained with H&E and for BrdU, P-Akt, P-β-catenin552, and alcian blue. Arrows indicate positively stained cells within crypt bases and arrow heads indicate staining in the mid-upper crypt regions. (B) Infection resulted in crypt lengthening as (C) proliferation (BrdU incorporation) increased 6-fold, as well as increased PI3K signaling determined by enumeration of (D) P-Akt+ epithelial cells/crypt and (E) P-β-catenin552+ IEC/crypt. (F) Enumeration of alcian blue+ cells/crypt demonstrates progressive loss of goblet cells coinciding with increased PI3K signaling to Day 14. Goblet cells re-populate crypts as PI3K signal returns to baseline. Values are mean ± SEM. Mag 200X with 400X inserts. Scale bar = 100 µm. *p ≤ 0.02, #p ≤ 0.01 vs. Day 0. Figure 2. PI3K inhibition impairs C. rodentium-induced Akt and β-catenin signaling and attenuates epithelial proliferation. Distal colon was removed from B6 mice 14 days after oral infection with C. rodentium treated with either DMSO (vehicle control, middle panels) or LY294002 (right panels). Left panels represent uninfected mice treated with LY294002. (A) Colonic tissue stained with H&E and for BrdU, P-Akt, P-β-catenin552, and alcian blue. Arrows indicate positively stained cells within crypt bases and arrow heads indicate staining in the mid-upper crypt regions. (B-F) LY294002 treatment alone had no effect relative to baseline, but impaired C. rodentium-induced crypt lengthening, PI3K signaling, proliferation, and goblet cell depletion. Values are mean ± SEM. Mag 200X with 400X inserts. Scale bar = 100 µm. *p < 0.01. Figure 3. PI3K inhibition does not affect epithelial apoptosis. (A) Distal colon sections from mice infected 14 days earlier with C. rodentium and treated with either DMSO or LY294002 were stained for apoptotic cells by the TUNEL technique (see methods). (B) Enumeration of TUNEL+ epithelial cells per 400X field. Values are mean ± SEM. Figure 4. Inhibition of PI3K signaling reduces β-catenin signaling in C. rodentium colitis. Colon sections from uninfected (day 0) and C. rodentium infected mice treated with DMSO or LY294002 (day 14) were processed and stained to assess (A) c-Myc (green) expression and (B) cyclinD1 (brown) expression. (C) Enumeration of c-Myc and cyclinD1 positive epithelial cells per crypt. Values are mean ± SEM. Mag 200X with 400X inserts. Figure 5. Inhibition of PI3K signaling increases and prolongs weight loss. C. rodentium infection caused transient weight loss at day 4 with recovery by day 7 and subsequent weight gain. Blockade of PI3K signaling worsened weight loss that did not recover until day 19 and was significantly reduced until day 28. *p < 0.01 vs. C. rodentium. (n=4-8 mice for each group). Figure 6. PI3K signaling is required for clearance of C. rodentium. In vivo colonization and clearance dynamics were monitored from day 7 after inoculation with C. rodentium to day 28. (A) Mice receiving LY294002 showed prolonged colonization within the colon and shedding of bacteria in the stool demonstrating PI3K inhibition reduces the ability to clear C. rodentium infection. (B) PI3K inhibition resulted in persistence of systemic infection within the spleen. Arrows indicate time points for LY294002 injection. *p < 0.05 vs. C. rodentium. (n=3-4 mice for each group, representative of 3 separate experiments). Figure 7. Interruption of PI3K signaling disrupts the epithelial barrier. (A) Top panels show two separate representative distal colons stained with H&E demonstrating expected crypt hyperplasia with relatively intact epithelial lining 14 days after infection with C. rodentium. Bottom panels show two separate representative distal colons in mice infected with C.

rodentium and treated with LY294002. PI3K inhibition of host epithelial responses resulted in


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increased disruption of the epithelial barrier and frank ulceration. Arrows indicate ulcer edges. Mag 100X. (B) Histologic scores for ulceration, inflammatory cell infiltration, and edema were assessed in colonic tissue from infected mice treated with DMSO or LY294002. Figure 8. PI3K is dispensable for adaptive and innate immune responses against C. rodentium. (A) Time course of mRNA (adaptive immunity) isolated from distal colon after C. rodentium infection alone, or with LY294002 treatment. (B) Distal colon segments were removed 14 days after infection, with and without LY294002 treatment, washed, and cultured overnight for direct cytokine analysis by ELISA corresponding to 8A mRNA. (C) Time course of mRNA (innate immunity) isolated from distal colon after C. rodentium infection alone, or with LY294002 treatment. (n=3-4 mice per group). Figure 9. PI3K inhibition does not reduce IL-22 production. PI3K inhibition does not affect either (A) mRNA or (B) colon explant culture ELISA measurement of IL-22 production 4 and 7 days after infection with C. rodentium. Figure 10. PI3K inhibition with wortmannin (WM) impairs host defense to C. rodentium. Distal colon was removed from B6 mice 14 or 18 days after oral infection with C. rodentium treated with either DMSO (vehicle control) or 0.1 mg/kg WM (methods). (A) WM treatment alone had no effect relative to baseline, but impaired C. rodentium-induced crypt lengthening. (B) C.

rodentium infection caused transient weight loss with recovery by day 7 and subsequent weight gain. Blockade of PI3K signaling worsened weight loss that did not begin to recover until day 16. (C) Mice receiving WM showed prolonged colonization within the colon and shedding of bacteria in the stool as well as in persistence of systemic infection within the spleen demonstrating PI3K inhibition reduces the ability to clear C. rodentium infection. (D) mRNA levels at day 14 and 18. Values are mean ± SEM. *p < 0.05 vs. C. rodentium + DMSO. #p < 0.01 vs. day 14. (n=5-8 mice per group).


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/cry

pt

#

#

##

C

0 7 1421 28

Day

0

1

2

3

4

5

P-β

-cate

nin

+/c

ryp

t

*

#

E

0 7 14 21 28

Day

0

1

2

3

4

5

P-A

kt+

/cry

pt

*

#

D

0 7 1421 28Day

F

Alc

ian

blu

e+

/cry

pt

0

4

8

12

16

20

*#


.org/D

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AC

. ro

den

tium

C. r

od

en

tium

+L

Y294002

BDE

C. rodC

. rod+LY

CF

BrdUP-AktP-β-cateninAlcian blue

LY

294002

Alcian blue+/crypt

0 3 6 9

12

15

LY

H&E

Crypt length (um)

0

100

200

300

400

*

010

20

30

40

50

60

70

BrdU+/crypt

*

0 1 2 3 4 5 6 7 8 9

P-Akt+/crypt

*

0 1 2 3 4 5 6 7

P-β-catenin+/crypt

*


.org/D

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C. rodentium C. rodentium + LY294002

0

1

2

3

4

5

C. r

od C. r

od

+ LY294002

TU

NE

L+

/HP

FA B


.org/D

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Uninfected C. rodentium C. rodentium

+ LY294002

Uninfected C. rodentium C. rodentium

+ LY294002

A

B

C

0

4

8

12

16

c-M

yc+

/cry

pt

0

10

20

30

40

cyclin

D1

+/c

ryp

t

Uninf

C. r

od

C. r

od

+ LY294002

Uninf

C. r

od

C. r

od

+ LY294002


.org/D

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-6-4-202468

1012

% c

han

ge b

od

y w

eig

ht

4 7 10 13 16 19 22 28

Day

0**

***

C. rodentium

C. rodentium + LY294002

Uninfected

LY294002


.org/D

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C. rodentium

C. rodentium + LY294002

10

102

103

104

105

7 14 18 21 28

Lo

g C

FU

/sp

leen

0

Day

A

B

1

10102

103

104

105

106

107

108

Lo

g C

FU

/co

lon

1010

109

7 14 18 21 280Day

1

106

*

*

*


.org/D

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C. ro

dC

. ro

d +

LY

294002

0

2

4

6

8

10

Ulceration

InfiltrationEdema

C. r

oden

tium

C. r

oden

tium

+ LY294002

A

B p = NS


.org/D

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pg

/mL

0

100

200

300

400IFN-γ

0

50

100

150

4 7 14 18 21 28

fold

-in

cre

ase

A B

C. ro

d

+ LY

Unin

fect

ed

C. ro

d

2500

0

500

1000

1500

2000

pg

/mL

0

20

40

60

80p

g/m

L

050

100150200250300

pg

/mL

TNF

fold

-in

cre

ase

0

1

2

3

4

5

4 7 14 18 21 28

IL-17A

fold

-in

cre

ase

0

20

40

60

80

4 7 14 18 21 28

CXCL9

fold

-in

cre

ase

0

20

40

60

80

100

4 7 14 18 21 28

Day 14

Day


0

20

40

60

80

4 7 14 18 21 28

fold

in

cre

ase Reg3γ

0

100

200

300

400

4 7 14 18 21 28

fold

in

cre

ase MMP-7

C


.org/D

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02040

6080

100120

140160

Day 4 Day 7

pg

/mL

mR

NA

fo

ld in

du

cti

on

0

500

1000

1500

2000

2500

3000

Day 4 Day 7

A B


.org/D

ownloaded from

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0

50

100

150

200

250

Cry

pt

len

gth

(u

m)

C. ro

d +

DM

SO

C. ro

d +

0.1

WM

0.1

WM

A

-20

-15

-10

-5

0

5

10

15

0 4 7 10 13 16 18

0.1 WM

C. rodentium + DMSO

C. rodentium + 0.1 WM

Day

% c

han

ge i

n b

od

y w

eig

ht

B

10

103

105

107

109

Day 14 Day 18

Lo

g C

FU

/co

lon

10

102

103

104

Day 14 Day 18

Lo

g C

FU

/sp

leen

C. rodentium + DMSO C. rodentium + 0.1 WMC

05

100

200

300

Day 14 Day 18

fold

-in

cre

as

e

IFNγ

0.1 WM

C. rodentium + DMSO

C. rodentium + 0.1 WM

0

20

40

60

80

Day 14 Day 18

iNOS

fold

-in

cre

as

e

0

100

200

300

Day 14 Day 18

CXCL9

fold

-in

cre

as

e

D

*

#

*

*

***


.org/D

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Downloaded from on May 9, 2020 by guest · 131 (Sigma, St. Louis, MO) dissolved in DMSO diluted 1:5...

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