Epithelial Tyrosine Phosphatase SHP-2 Protects against IntestinalInflammation in Mice

Geneviève Coulombe,a Caroline Leblanc,a Sébastien Cagnol,a Faiza Maloum,a Étienne Lemieux,a Nathalie Perreault,a

Gen-Sheng Feng,b François Boudreau,a Nathalie Rivarda

Département d’Anatomie et de Biologie Cellulaire, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec, Canadaa;Department of Pathology and Division of Biological Sciences, University of California San Diego, La Jolla, California, USAb

Polymorphisms of PTPN11 encoding SHP-2 are biomarkers for ulcerative colitis (UC) susceptibility. However, their functionalrelevance is unknown. We thus investigated the role of epithelial SHP-2 in the control of intestinal homeostasis. Mice with anintestinal epithelial cell-specific SHP-2 deletion (SHP-2IEC-KO mice) were generated. Control and SHP-2IEC-KO mice were moni-tored for clinical symptoms and sacrificed for histological staining and Western blot analyses. Cytokines and chemokines, aswell as intestinal permeability, were quantified. SHP-2 mRNA expression was evaluated in control and UC patients. SHP-2IEC-KO

mice showed growth retardation compared to control littermates and rapidly developed severe colitis. Colon architecture wasmarkedly altered with infiltration of immune cells, crypt abscesses, neutrophil accumulation, and reduced goblet cell numbers.Decreased expression of claudins was associated with enhanced intestinal permeability in mutant SHP-2IEC-KO mice. Inflamma-tory transcription factors Stat3 and NF-�B were hyperactivated early in the mutant colonic epithelium. Levels of several epithe-lial chemokines and cytokines were markedly enhanced in SHP-2IEC-KO mice. Of note, antibiotic treatment remarkably impairedthe development of colitis in SHP-2IEC-KO mice. Finally, SHP-2 mRNA levels were significantly reduced in intestinal biopsy speci-mens from UC patients. Our results establish intestinal epithelial SHP-2 as a critical determinant for prevention of gutinflammation.

Crohn’s disease (CD) and ulcerative colitis (UC) involve inter-actions between the immune system, genetic susceptibility,

and the environment (1). Genetic studies show that CD and UCare polygenic diseases with strong environmental influences. Inthis regard, a wealth of genes associated with CD and UC havebeen uncovered. For this matter, animal models have providedfundamental insights into the importance of immunologic dys-regulation and intestinal microbiota composition (2). Notably,epithelial barrier function is critical to gut homeostasis (3). Anearly demonstration of this concept was the increased intestinalinflammation observed in mice with mucosal leakiness caused bydominant-negative epithelial N-cadherin (4). The barrier conceptis further supported by the spontaneous colitis in mice lacking themajor component of intestinal mucus, Muc2 (5). In addition,mice with intestinal epithelial cell (IEC)-specific ablation ofNF-�B components fail to control the luminal microbial flora,and inflammation is triggered by an exaggerated immune re-sponse to invading bacteria (6).

Phosphoprotein tyrosine phosphatases (PTPs) have beenlinked to intestinal inflammation. PTPN2 gene variants are asso-ciated with susceptibility to both CD and UC (7). Heterozygousdeletion of PTPN2, coding for T cell PTP (TCPTP), led to in-creased susceptibility to dextran sulfate-mediated intestinal in-flammation (8). Polymorphisms in the PTPN11 gene coding forSHP-2 (Src homology 2-containing protein tyrosine phosphatase)were also associated with UC susceptibility (9). Additional single-nucleotide polymorphisms in close proximity to the PTPN11 lo-cus were also identified in CD (10), suggesting that SHP-2 mightplay a role in intestinal inflammation.

SHP-2 is an Src homology 2-containing PTP expressed in mostembryonic and adult tissues. SHP-2 regulates many cellular func-tions, including progenitor cell development, cellular growth, tis-sue inflammation, cellular chemotaxis, and cell survival (11, 12).

SHP-2 is positively required for the functions of several growthfactors and metabolic pathways, having far-reaching implicationsfor disease pathways and disorders, such as diabetes, neurodegen-eration, and cancer (13).

Here, we investigated the specific role of intestinal epithelialSHP-2 by generating mice lacking SHP-2 expression in IECs.Remarkably, mice develop spontaneous intestinal inflamma-tion with clinical and histopathological features strikingly sim-ilar to UC.

MATERIALS AND METHODSAnimals. Shp2flox/flox mice (F3) (14) were backcrossed with C57BL/6 micefor nine generations. Experiments were performed with F5 to F12 mice.The C57BL/6 12.4KbVilCre transgenic line was provided by D. Gumucio(University of Michigan, Ann Arbor, MI) (15). Mutations were genotypedas described previously (14, 15). Experiments were approved by the Ani-mal Research Ethic Committee of the Université de Sherbrooke.

Western blot analysis. Proteins were isolated from total colon (neo-nates), from scraped mucosa (2-week-old mice), or from cultured cells inchilled RIPA buffer, and Western blot analyses were performed as de-scribed previously (16). Antibodies against SHP-2 and extracellular sig-nal-regulated kinase 1 and 2 (ERK1/2) (Santa Cruz Biotechnology, SantaCruz, CA); phosphorylated RelA (S536), RelA, Stat3, phosphorylatedStat3 (Y705), and phosphorylated ERK1/2 (T202/Y204) (Cell Signaling,Danvers, MA); claudins (Invitrogen, Burlington, ON, Canada); E-cad-

Received 10 January 2013 Returned for modification 28 January 2013Accepted 20 March 2013

Published ahead of print 25 March 2013

Address correspondence to Nathalie Rivard, Nathalie.Rivard@USherbrooke.ca.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/MCB.00043-13

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herin (BD Biosciences, Mississauga, ON, Canada); and �-actin (Milli-pore, Billerica, MA) were used for Western blot analyses. Horseradishperoxidase–anti-mouse and –anti-rabbit antibodies were purchased fromAmersham Biosciences (Pittsburg, PA). Densitometric analyses were per-formed using Image J software (developed by the U.S. National Institutesof Health) and were carried out on some Western blots. The relativeactivated levels of Stat3, RelA, or Erk were established with the ratios ofphosphorylated Stat3/total Stat3 or �-actin, phosphorylated RelA/totalRelA or �-actin, or phosphorylated Erk1/2 to total Erk1/2 or �-actin,which were in turn compared to those of control mice, which were set at 1.

Histological staining and immunohistochemistry. Colons werefixed, sectioned, and stained as described previously (16).

Immunofluorescence. SHP-2IEC-KO and control murine colons werefixed in 4% paraformaldehyde overnight at 4°C and then dehydrated andembedded in paraffin. Sections (5 �m) were applied to Probe-On Plusslides (Fisher Scientific Canada). An immunofluorescence assay againstphosphorylated Stat3 (Y705; Cell Signaling) was performed as previouslydescribed (17), except that 2% phosphate-buffered saline– bovine serumalbumin (PBS-BSA) 2% was used.

DAI. Mice were scored based on a scale of 0 to 4 for stool consistency,rectal bleeding, and blood loss, as well as colon hardness, and a cumulativedisease activity index (DAI) was calculated, as described previously (18).

Electron microscopy. Tissues intended for electron microscopy anal-ysis were fixed with 2.8% glutaraldehyde in 0.1 M cacodylate buffer andprocessed as described previously (19).

RNA isolation and quantitative real-time PCR (qPCR). RNA wasisolated from the scraped colonic mucosa of 2-week-old mice or fromIEC-6 cells using the RNeasy minikit (Qiagen). Reverse transcription andquantitative PCR were performed as previously described (20) or wereperformed by the RNomics Platform at the Université de Sherbrooke(Sherbrooke, QC, Canada). Target expression was quantified relative toGAPDH expression in mice and to Tubb5 expression in cells. All primersequences and cycling conditions are available upon request.

Intestinal permeability in vivo. Permeability was examined using thefluorescein isothiocyanate (FITC)-labeled dextran method. Mice wereforce fed with 60 mg/100 g body weight of FITC-dextran (Sigma-Aldrich,Oakville, ON, Canada) 4 h before sacrifice. The FITC concentration in theserum was determined by using a BioTek Synergy HT (480/520-nm) platereader (Winooski, VT).

Cytokine and chemokine assays. Production of cytokines andchemokines was determined with an antibody array (AAM-INF-G1-8;RayBiotech, Inc., Norcross, GA). The array was scanned with the Cy3channel using a ScanArray Express dual-color confocal laser scanner(PerkinElmer, Woodbridge, ON, Canada). The mean level of each cyto-kine/chemokine produced in six SHP-2-deficient mice was calculated rel-ative to the mean level observed in six control mice, which was set at 1.

Antibiotic treatments. Mice were treated during pregnancy and lac-tation periods with a cocktail of antibiotics (all from Sigma-Aldrich),including ampicillin (1 g/liter), vancomycin (500 mg/liter), streptomycin(450 mg/liter), and metronidazole (1 g/liter), with saccharin (1%) addedto the drinking water. At weaning, antibiotics were added to the drinkingwater of the offspring and were changed every 2 days. In a second group,antibiotic treatment began at weaning. For both groups, mice were sacri-ficed at the age of 9 weeks, and DAIs were calculated.

Generation of shRNA, lentivirus production, and cell infection. Thelentiviral short hairpin RNA (shRNA) expression vector (pLenti6-U6)was constructed as described previously (21). The sequences of rat andhuman shRNA oligonucleotides are available upon request. IrrelevantpLenti-shGFP and pLenti-shRNA with the scrambled sequence of humanSHP-2 short hairpin RNA (shSHP-2) were used as negative controls(shControl). Lentiviruses produced in 293T cells were used for infectionof IEC-6 or Caco-2/15 cells according to Invitrogen recommendations.

Cell culture. The human colon cell line Caco-2/15, obtained fromAndrea Quaroni (Cornell University, Ithaca, NY), and the rat intesti-nal epithelial cell line IEC-6, provided by the ATCC (Manassas, VA),were cultured as described previously (22). IEC-6 cells were serumstarved for 24 h and then treated with 10 ng/ml of interleukin 6 (IL-6)(Bioshop, Burlington, ON, Canada) or 12.5 �g/ml of lipopolysaccha-ride (LPS) (serotype O111:B4; Enzo Life Sciences, Farmingdale, NY).

TEER. Transepithelial electric resistance (TEER) was measured withan epithelial voltohmmeter (EVOM2; World Precision Instruments,Sarasota, FL) on shControl and shSHP-2 Caco-2/15 cells or on 10-day-postconfluent, differentiated Caco-2/15 cells grown on Transwell 0.4-�mfilters (Corning, Cambridge, MA) treated or not for 24 h with 5 �M or 20�M MEK inhibitor U0126 (L.C. Laboratories, Woburn, MA). TEER wascalculated as ohms multiplied by cm2 of cell surface after subtracting theresistance values measured in cell-free inserts.

SHP-2 gene transcripts in UC patients. SHP-2 mRNA transcriptswere analyzed in tissue samples from patients with chronic and active UCusing TissueScan Real-Time arrays purchased from Origene Technologies(Rockville, MD). According to instructions from Origene Technologies,biopsy specimens were characterized histologically. Samples composed ofat least 40% epithelial mucosa were analyzed. Control and UC sampleswere collected from different individuals. After resuspension of lyophi-lized cDNAs, qPCR was performed and normalized against �-actin. ThePCR conditions and primers used are available upon request.

Statistical analysis. Statistical analyses were done using the Studenttwo-tailed t test calculated with GraphPad (Irvine, CA) Prism 5 softwarefor all experiments except patient analyses, which were analyzed by theMann-Whitney test. Differences were considered significant at P values of

FIG 1 Epithelial SHP-2 deletion results in growth retardation. (A) Westernblot analysis of SHP-2 protein expression along the rostrocaudal axis frommucosal enrichments of duodenum, jejunum, ileum, and colon. �-Actin wasused as the loading control. (B) Mucosal enrichments from SHP-2IEC-KO andcontrol jejunums and colons were analyzed by Western blotting for SHP-2 and�-actin expression. (C) Immunohistochemistry of SHP-2 protein in 2-week-old murine colonic tissues. Scale bars, 50 �m. (D) Body weights of SHP-2IEC-KO mice and control littermates at different ages (n � 12 per group). ***,P � 0.001. The error bars indicate the SEM. (E) Representative photographs of1-month-old control and SHP-2IEC-KO mice.

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�0.05, �0.01, or �0.001. Error bars indicate the standard errors of themean (SEM).

RESULTSSHP-2IEC-KO mice display growth retardation. We gener-ated mice lacking SHP-2 expression specifically in IECs(SHP-2IEC-KO) by crossing mice homozygous for the floxedexon 4 of the Shp-2 gene (Shp2flox/flox) (14) with villin-Cretransgenic mice, expressing the transgene around embryonicday 15.5 (E15.5) in IECs (15, 23). Western blot analysis ofextracts from mucosal enrichments confirmed SHP-2 expres-sion throughout the rostrocaudal axis in control mice (Fig. 1A)and the loss of SHP-2 expression in the small and large intes-tinal epithelium in SHP-2IEC-KO mice (Fig. 1B and C).

SHP-2IEC-KO mice were born at the expected Mendelian ratio,and no difference in body weight was observed at birth. However,3 weeks after birth, and more significantly at 4 weeks, SHP-2IEC-KO

mice exhibited growth retardation in comparison to control lit-termates (Fig. 1D). Furthermore, 1 month after birth, SHP-2IEC-KO mice were leaner (Fig. 1E), and their body mass index wassignificantly reduced (by 30.8%) in comparison to controls. Nodifference in body weight was observed between heterozygous andcontrol littermates (data not shown).

SHP-2IEC-KO mice spontaneously develop severe colitis. Onemonth after birth, SHP-2IEC-KO mice showed diarrhea and rectalbleeding with higher mortality than control mice (Fig. 2A). Asshown in Fig. 2B, the DAI was significantly higher in SHP-2IEC-KO

mice than in control littermates. Macroscopic examination re-vealed severe pancolitis affecting all parts of the colon (Fig. 2C).No inflammation was observed in the small intestine, and no in-flammation was observed in colons from heterozygous mice.

Histological analysis demonstrated that the colons of1-month-old SHP-2IEC-KO mice exhibited signs of colitis, includ-ing immune cell infiltration, longer crypts, and apparent reduc-tion of goblet cells. The distal colon was more severely affected 1month after birth, with the presence of crypt abscesses, enlargedcrypts, and marked infiltration of mononuclear cells into the mu-cosa and submucosa (Fig. 2D). H&E staining of a 3-month-oldSHP-2IEC-KO murine colon fixed in a Swiss roll position revealedinflammation from the rectum to the colon (data not shown).Importantly, the intestinal mucosa of newborn mutant mice ex-hibited no significant alteration in IEC survival (data not shown),mucosal histology, or mucin expression (Fig. 3A). In contrast,colons of 2-week-old (Fig. 3B) and 4-week-old (Fig. 2D) SHP-2IEC-KO mice exhibited marked reductions in mucin staining andgoblet cell numbers (Fig. 4A). qPCR analyses of goblet cell mark-ers, mucin 2 (Muc2) and trefoil factor 3 (Tff3), revealed no differ-ence in neonate littermates (Fig. 4B), while significant reductionsof Muc2 (51.6%) and Tff3 (35.9%) expression were observed 2weeks after birth (Fig. 4C). Notably, the remaining goblet cellspresented large vacuoles filled with mucus, similar to mature gob-let cells from controls (Fig. 4D).

To assess the expression of modulators of the immune re-

FIG 2 SHP-2IEC-KO mice spontaneously develop colitis. (A) Survival rates of SHP-2IEC-KO mice versus control littermates (n � 16 per group). (B) DAIs of1-month-old SHP-2IEC-KO and control littermates were calculated by scoring stool softness, occult fecal blood, rectal bleeding, and colon hardness (n � 15 pergroup). ***, P � 0.001. The error bars indicate the SEM. The horizontal bar indicates the mean value. (C) Representative photographs of 4-month-old controland SHP-2IEC-KO murine colons. (D) H&E, alcian blue, and immunohistochemistry of MPO staining from 1-month-old control and SHP-2IEC-KO murinecolons. Asterisks, crypt abscesses; brackets, longer crypts; arrows, immune infiltration. Scale bars, 100 �m.

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sponse, a cytokine antibody array assay was performed on colonprotein extracts. This analysis identified several cytokines andchemokines that were significantly upregulated in SHP-2IEC-KO

mice (Table 1), while others remained unaffected (Table 2). Ofnote, the epithelial chemokines CXCL1 and CXCL5 and the Th2-associated chemoattractant CCL11 were markedly enhanced in2-week-old SHP-2IEC-KO mice. This upregulation of chemokineswas correlated with the increased detection of the myeloperoxi-dase (MPO) signal associated with leukocytes attracted by thesame chemokines released at the inflammation site (Fig. 2D). In3-week-old mice, IL-1�, IL-1�, IL-6, and IL-12 levels were signif-icantly enhanced, in contrast to tumor necrosis factor alpha(TNF-�) and gamma interferon (IFN-�).

Loss of IEC-specific SHP-2 deregulates intestinal permeabil-ity and barrier components. Since intestinal epithelial barrier dis-ruption represents an important feature of inflammatory boweldisease (IBD) (3), we measured intestinal permeability and theexpression of junctional proteins. As illustrated in Fig. 5A, intes-tinal permeability was markedly increased in both 2-week- and1-month-old SHP-2IEC-KO mice compared to control littermates.Protein levels of occludin (decreased 30%) (data not shown) andclaudins 1 (decreased 63%), 4 (decreased 63%), 8 (decreased

69%), and 15 (decreased 61%) were significantly decreased in2-week-old SHP-2IEC-KO colonic epithelium, while E-cadherinand claudin 2 and 3 levels were not modulated (Fig. 5B). Analysisof claudin expression has also been done in neonate colons, with ageneral trend of reduced expression in SHP-2IEC-KO mice (datanot shown). No significant difference in the expression of junc-tional proteins was noted in the small intestines of mutant mice(data not shown).

To determine the cell-intrinsic effect of SHP-2 ablation on per-meability, we generated lentiviruses encoding anti-shSHP-2 tosuppress SHP-2 expression in Caco-2/15 cells (Fig. 5C). This co-lonic epithelial cell line forms apical junctional complexes, result-ing in a polarized monolayer postconfluence, with increasedTEER. Importantly, SHP-2 silencing in Caco-2/15 cells compro-mised barrier function, as evidenced by the significant decrease intheir TEER in comparison to control cells (Fig. 5D).

Loss of IEC-specific SHP-2 deregulates epithelial ERK, Stat3,and NF-�B signaling pathways. SHP-2 regulates mitogen-acti-vated protein (MAP) kinase, JAK/Stat, and NF-�B signaling path-ways (24, 25). Western blot analysis of colonic epithelial enrich-ments from 2-week-old mice showed that ERK1/2phosphorylation was reduced by 4.26-fold (�1.282-fold; n � 3)(Fig. 6A), whereas p38 and JNK MAP kinase phosphorylation wasnot altered (data not shown). In addition, the transcription factorsStat3 (Y705) and NF-�B (RelA; S536) were hyperphosphorylated(activated) by 4.72-fold (�1.078-fold; n � 3) and 1.70-fold(�0.321-fold; n � 3), respectively, in the colonic epithelia of2-week-old SHP-2IEC-KO mice in comparison to controls (Fig. 6Band C). In neonates, while ERK1/2 and RelA phosphorylationlevels varied between individuals, hyperphosphorylated Stat3 wasconsistently detected in all mutant mice analyzed (Fig. 6D to F).Immunofluorescence confirmed increased Stat3 phosphorylationin colonic epithelium from SHP-2IEC-KO mice (Fig. 6G). Consis-tent with these results, SHP-2 silencing in cultured IECs increasedlevels of phosphorylated Stat3 under both unstimulated (6.61-fold) and stimulated (IL-6; 4.29-fold after 5 min, 2.89-fold after 30min) conditions. Furthermore, SHP-2-depleted cells were unableto induce ERK1/2 phosphorylation in response to IL-6 (Fig. 6H).

Modulation of ERK signaling has been associated with bothdisruption and maintenance of barrier function in other epithelialcell contexts (26–31). Therefore, we next evaluated whether theinhibition of ERK activity found in SHP-2-deficient IECs couldaccount for the increased permeability observed. We treatedCaco-2/15 monolayers with the specific MEK inhibitor U0126 andobserved that ERK inhibition did not decrease basal TEER. Incontrast, TEER was significantly increased following U0126 treat-ment (Fig. 7A and B), suggesting that MEK/ERK negatively, notpositively, controls barrier function in these cells. Thus, this resultindicates that ERK inactivation was not involved in the inhibitionof TEER observed following SHP-2 silencing in IECs.

Antibiotic treatment attenuates the development of colitis inSHP-2IEC-KO mice. To evaluate the role of the microbiota in theonset of inflammation (2, 32), SHP-2-deficient mice and controllittermates were treated with a cocktail of antibiotics previouslyreported to inhibit gut microflora proliferation (33). In the firstgroup, mice were treated during pregnancy and lactation periods.At weaning, antibiotics were added to the drinking water of theoffspring. In the second group, antibiotic treatment began atweaning. For both groups, mice were sacrificed at the age of 9weeks and DAIs were calculated. As shown in Fig. 8A and B, both

FIG 3 Intestinal histology in young SHP-2IEC-KO mice. H&E and alcian bluestainings were performed on 1-day-old (A) and 2-week-old (B) control andSHP-2IEC-KO murine colons. Scale bars, 100 �m.

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antibiotic treatments significantly inhibited the development ofcolitis in SHP-2IEC-KO mice, as shown by the marked reduction ofthe DAI and the maintenance of some histological integrity. Thus,the beneficial effect of antibiotics on the DAI highlights the con-tribution of microflora to the initiation of inflammation in SHP-2-deficient mice. Interestingly, SHP-2-deficient IECs in culturewere hyperresponsive to LPS stimulation, as visualized by themarked increase in RelA phosphorylation (6.42-fold after 5 min;2.64-fold after 30 min) and expression of Cxcl1 and Cxcl5 chemo-kine genes (Fig. 8C and D).

TABLE 1 Altered production of chemokines and cytokines inSHP-2IEC-KO mice compared to control littermates

Cytokine, chemokine,or other

Fold production in SHP-2IEC-KO micea

2 wk 3 wk

IFN-� 0.96 0.98IL-1� 0.98 4.43c

IL-1� 0.97 1.36c

IL-2 0.84b 0.80b

IL-4 0.91b 0.95IL-6 0.92b 3.43b

IL-10 0.87c 0.84b

IL-12 1.07 1.32b

IL-17 0.87b 1.07TNF-� 0.88b 0.89c

CCL1 (TCA-3) 0.81c 0.71c

CCL2 (MCP-1) 0.98 9.11c

CCL3 (MIP-�) 0.89c 1.23CCL5 (RANTES) 1.76b 2.75c

CCL9 (MIP-�) 1.31b 1.26b

CCL11 (eotaxin-1) 1.18b 5.10d

CCL25 (TECK) 0.90b 0.87b

CXCL1 (KC) 2.28 14.1c

CXCL5 (LIX) 3.39b 8.94d

CXCL9 (MIG) 0.92b 1.24b

CXCL13 (BLC) 1.13 3.25d

CX3CL1 (fractalkin) 0.86c 0.73c

XCL1 (lymphotactin) 0.92b 0.92c

G-CSFe 0.96 3.29b

TIMP-1 1.70c 3.51d

sTNFR II 1.45 2.05b

a Compared to control littermates (n � 6 mice per group).b P � 0.05.c P � 0.01.d P � 0.001.e G-CSF, granulocyte colony-stimulating factor.

FIG 4 Goblet cells in SHP-2IEC-KO mice. (A) Epithelial cells from 2-week-old murine distal colons stained positively for alcian blue were counted in 15 crypts(n � 3 per group). ***, P � 0.001. The error bars indicate the SEM. (B and C) qPCR of Muc2 and Tff3 mRNAs in total extracts from 3-day-old (B) and in enrichedmucosal extracts from 2-week-old (C) SHP-2IEC-KO mice versus controls. Relative Muc2 and Tff3 expression was normalized with the housekeeping gene GAPDH(n � 4 per group). *, P � 0.05. The error bars indicate the SEM. (D) Electron microscopy shows similar maturation levels of goblet cells present in colons ofcontrol and SHP-2IEC-KO mice. Scale bars, 2 �m.

TABLE 2 Chemokines and cytokines not significantly altered in SHP-2IEC-KO mice compared to control littermates

Cytokine, chemokine,or othera

Fold production in SHP-2IEC-KO miceb

2 wk 3 wk

IL-3 0.96 0.98IL-9 0.94 0.85IL-13 1.00 1.04CCL24 (eotaxin-2) 1.00 1.05CXCL11 (I-TAC) 0.98 1.08CXCL12 (SDF-1) 1.05 1.13CD30L 0.95 0.97Fas ligand 0.91 0.99GM-CSF 0.92 0.93Leptin 0.98 1.13M-CSF 0.91 0.98TIMP-2 0.96 1.01sTNFR I 1.14 1.23a GM-CSF, granulocyte-macrophage colony-stimulating factor; M-CSF, macrophagecolony-stimulating factor.b Compared to control littermates (n � 6 mice per group).

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Decreased mRNA expression of SHP-2 in UC patients. Theexpression status of SHP-2 in UC patients was assessed by qRT-PCR. As shown in Fig. 9, SHP-2 gene transcripts were significantlyreduced in intestinal biopsy specimens from patients with chronicand active UC compared to controls. These results emphasize theinverse relationship between SHP-2 expression and the intestinalinflammatory phenotype.

DISCUSSION

IBDs are a result of genetic susceptibility, defects in host immu-nity, intestinal microflora, and other environmental factors (1).Genetic studies have identified IBD susceptibility genes, such asthe SHP-2-encoding PTPN11 gene, associated with intronic poly-morphisms first described in Japanese UC patients (9). Since wefound that SHP-2 gene transcripts were significantly reduced inpatients with active UC, this could suggest that PTPN11 intronicpolymorphisms affect SHP-2 expression. However, additional

biochemical and molecular studies in a large number of humanspecimens will be needed to verify this hypothesis.

Here, we investigated the role of epithelial SHP-2 in the controlof intestinal inflammatory homeostasis. We found that mice withan IEC-specific deletion of SHP-2 develop intestinal inflamma-tion with clinical and histopathological features strikingly similarto UC. Chemokines and cytokine secretion profiles indicated thatthe immunophenotype of SHP-2IEC-KO mice was mostly similar tothe immunophenotype observed in patients suffering from UC asopposed to CD, a prototypical Th1 condition mediated by theIL-12–IFN-�–TNF-� cytokine axis (34). Accordingly, colon ar-chitecture was markedly altered, with immune cell infiltration,longer crypts, increased permeability, abscesses, and neutrophilaccumulation. In addition, we found that a reduction of goblet cellnumbers in the SHP-2-deficient murine colon is one of the earliesthistologic modifications. Accordingly, while CD is associated withgoblet cell increases and thicker mucus layers (35), UC is charac-terized by a reduction in goblet cells (3) and secreted mucus (36).Although it remains unclear whether UC changes in mucus pro-duction are causative of or secondary to inflammation (37), thelack of these changes in CD indicates that they are not a universalconsequence of intestinal inflammation. Furthermore, Muc2�/�

mice spontaneously develop colitis (5) through a diminished mu-cus barrier, which could lead to greater toxin and antigen expo-sure, triggering local injuries and mucosal inflammation. Thus,the reduction in goblet cell numbers and in protective factors,such as TFF3 and MUC2, could explain in part the spontaneouscolitis of SHP-2IEC-KO mice.

In addition to goblet cell decreases, SHP-2-deficient micedisplay a downregulation of claudins 1, 4, 8, and 15 in the colonbefore the appearance of histopathological manifestations. In-terestingly, claudins 1, 4, and 8 are considered to be part of thebarrier-building group of claudins known to decrease paracel-lular permeability (38). In contrast, expression of these clau-dins was not altered in the SHP-2-deficient murine ileum, sug-gesting different roles for SHP-2 in the small intestine and thecolon. Furthermore, each claudin showed a unique and distinctexpression pattern along the proximal-to-distal and the crypt-villus axes (39). In addition, claudin expression varies duringdevelopment, and barrier functions, such as permeability tosolutes and ions, are determined by the combination of the 24claudin species (40). These intricate differences in claudin ex-pression from one intestinal section to another may well ex-plain the specific SHP-2-dependent alterations in the colon, asopposed to the small intestine. Interestingly, the decrease inclaudin protein expression found in the colon may be attribut-able to protein degradation rather than transcriptional regula-tion, since claudin mRNA levels were not modified in SHP-2-deficient colons (data not shown). Importantly, degradation oftight-junction (TJ) proteins, including claudins, through en-docytosis is clearly involved in remodeling TJ complexes be-tween cells (41). Accordingly, increased intestinal permeabilitywas observed in SHP-2IEC-KO mice compared to controls. Inaddition, SHP-2 silencing in Caco-2/15 colonic epithelial cellsmarkedly decreased TEER, indicating that increased paracellu-lar permeability was intrinsic to specific SHP-2 IEC deletionand was not triggered by paracrine effects resulting from in-creased inflammatory infiltrates. However, determining howSHP-2 depletion in IECs affects TJ remodeling and permeabil-ity will require further experiments. Currently, experiments

FIG 5 Loss of IEC-specific SHP-2 deregulates intestinal epithelial perme-ability and barrier components. (A) Intestinal permeability was deter-mined in control and mutant mice (n � 10 per group) by measuring levelsof FITC-dextran 4 h after its administration by gavage. *, P � 0.05. Theerror bars indicate the SEM. (B) Mucosal enrichments from 2-week-oldSHP-2IEC-KO and control murine colons were analyzed by Western blottingfor the expression of selected junctional proteins. �-Actin served as theloading control. (C) Caco-2/15 cells were infected with either recombinantlentiviruses encoding an shRNA that specifically knocks down SHP-2(shSHP-2) or negative-control shRNA (shControl). After selection, Caco-2/15 cell populations were lysed to analyze SHP-2 protein expression byWestern blotting. �-Actin served as the loading control. (D) Caco-2/15shControl and shSHP-2 cells were grown on porous membranes, and TEERwas measured in triplicate at days 0, 6, 12, and 18 postconfluence. ***, P �0.001. The error bars indicate the SEM.

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are in progress to determine the tyrosine phosphorylation sta-tus of occludin and claudins, since this could alter TJ protein-protein interactions, disrupting TJ-actin cytoskeletal linkageand increasing permeability (42, 43).

To determine the molecular mechanisms of intestinal in-flammation resulting from SHP-2 depletion, we analyzed theactivation levels of SHP-2 signaling effectors. We found in-creased Stat3 and RelA phosphorylation levels and reducedERK1/2 phosphorylation before the appearance of colitis in2-week-old mice. This altered ERK signaling is reminiscent ofresults demonstrating that SHP-2 mediates ERK activation inresponse to several growth factors (24, 44). Additionally, weshowed here that SHP-2 is also required for ERK activation in

response to IL-6 and LPS. How altered ERK activation in SHP-2-deficient mice contributes to the inflammatory phenotyperemains unknown. Activation of ERK signaling usually pro-motes IEC proliferation (45). However, although decreasedphosphorylated ERK was detected in the SHP-2IEC-KO intes-tine, elongated crypts were consistently observed, suggestingthat proliferation was increased. Recently, ERK activation wasreported to be critical for chemokine-induced intestinalwound healing (46). Although not examined in our model, itremains possible that decreased ERK1/2 activation alters theability of epithelial cells to adhere to or rapidly migrate acrossthe remodeled matrix of the inflamed mucosa.

SHP-2 is intimately associated with Jak/Stat signaling pathways(47, 48). For example, SHP-2 diminishes the signal relayed fromgp130 (49, 50), the signal-transducing receptor subunit for IL-6.Mutation of the gp130 SHP-2/SOCS3-binding site blocks signaltransduction via the Ras/ERK/AP1-signaling cascade while recip-rocally increasing IL-6-mediated activation of JAK/Stat3 signal-ing. Consistent with those reports, we have shown that SHP-2-depleted IECs, stimulated with IL-6 or not, exhibited increasedphosphorylated Stat3. This is in line with the results of Lehmann etal. (49), who showed that the expression of dominant-negativeSHP-2 increases basal gp130, Jak1, and Stat3 phosphorylation,implying that the basal active Stat3 kinase Jak1 is negatively regu-lated by SHP-2. Paradoxically, however, conditional knockoutmice with IEC deletion of Stat3 exhibited more severe experimen-tally induced acute colitis than control animals (51, 52). Althoughthese studies demonstrate that epithelial Stat3 activation mediatesmucosal protective functions, other evidence suggests that Stat3hyperactivation may also contribute to inflammation. Indeed, in-creases in serum IL-6 concentrations and Stat3 hyperactivationwere observed in IBD patients, as well as in experimental colitis

FIG 6 Epithelial SHP-2 deletion deregulates signaling pathways. Mucosal enrichments from 2-week-old (A to C) and total extracts from newborn (D to F)SHP-2IEC-KO and control murine colons were analyzed by Western blotting for the expression of phosphorylated ERK1/2 (T202/Y204) (pERK) and ERK1/2,phosphorylated Stat3 (Y705) and Stat3, and phosphorylated RelA (S536) and RelA. �-Actin served as the loading control. (G) Immunofluorescence againstphosphorylated STAT3 (Y705) was performed on colonic tissue from 2-week-old control and SHP-2IEC-KO mice. Scale bars, 100 �m. (H) IEC-6 cell populationsstably expressing an shRNA that specifically knocks down SHP-2 (shSHP-2) or negative-control shRNA (shControl) were treated with IL-6 (10 ng/ml) and lysed.Expression of phosphorylated Stat3 (Y705), phosphorylated ERK1/2 (T202/Y204), SHP-2, and �-actin was analyzed by Western blotting.

FIG 7 Impact of ERK inhibition on Caco-2/15 barrier function. (A) Caco-2/15 cells were treated for 24 h with the MEK inhibitor U0126 (5 �M and 20�M), lysed, and analyzed by Western blotting for the expression of phosphor-ylated ERK1/2 (T202/Y204) and ERK1/2. �-Actin served as the loading con-trol. (B) Ten-day-postconfluence Caco-2/15 cells were treated for 24 h with theMEK inhibitor U0126 (5 �M and 20 �M), and TEER was measured in qua-druplicate. **, P � 0.01; ***, P � 0.001. The error bars indicate the SEM.

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models (53, 54). Likewise, Stat3 is hyperphosphorylated in bothcolonic epithelial cells and lamina propria mononuclear cells incolitis (55). Mouse colonization with the human commensal bac-terium enterotoxigenic Bacteroides fragilis activates Stat3 in co-lonic epithelial cells and in a subset of infiltrating immune cells,initiating a Th17-driven mucosal immune response and inflam-mation (55). In addition, growth hormone (GH) inhibits Stat3activation in IECs of IL-10-null mice, resulting in reduced colitissymptoms associated with increased SHP-2/gp130 binding (55).Therefore, future studies should determine whether epithelialStat3 activation protects from and/or contributes to the develop-ment of the inflammatory phenotype in SHP-2IEC-KO mice.

Multiple lines of evidence suggest that NF-�B in mucosal cells

actively contributes to the development and maintenance of in-testinal inflammation (56). Interestingly, SHP-2 closely regulatesNF-�B activity. For instance, IL-6 production during IL-1�/TNF-� stimulation of fibroblasts requires both SHP-2 activationand the ability of SHP-2 to promote NF-�B activity (25). SHP-2also plays a positive signaling role in NF-�B activation in macro-phages (57). In contrast, our data indicate that NF-�B signaling isnot altered in the intestinal epithelia of SHP-2-deficient neonatesor after SHP-2 silencing in cultured IECs. However, increasedphosphorylated RelA was found in epithelial extracts from2-week-old SHP-2IEC-KO mice. In addition, SHP-2-depleted cul-tured IECs exhibited increased NF-�B activation and chemokineproduction in response to the microbial antigen LPS. Thus, one

FIG 8 Reduced inflammation in SHP-2IEC-KO mice treated with antibiotics. (A) In the first group, mice were treated during pregnancy and lactationperiods with a cocktail of antibiotics, including ampicillin, vancomycin, streptomycin, and metronidazole, added to the drinking water. At weaning,antibiotics were added to the drinking water of the offspring [Control treated (newborn) and SHP-2IEC-KO treated (newborn)]. In the second group,antibiotic treatment began at weaning [Control treated (3 weeks) and SHP-2IEC-KO treated (3 weeks)]. For both groups, mice were sacrificed at the age of9 weeks, and DAIs were calculated by scoring stool softness, occult fecal blood, rectal bleeding, and colon hardness (n � 6 per group). *, P � 0.05; ***,P � 0.001. The error bars indicate the SEM. The horizontal bars indicate the mean value. (B) H&E staining was performed on distal colons of control andSHP-2IEC-KO mice treated (newborn or 3 weeks) or not with antibiotics. Scale bars, 100 �m. (C) IEC-6 cell populations stably expressing an shRNA thatspecifically knocks down SHP-2 (shSHP-2) or negative-control shRNA (shControl) were treated with LPS (12.5 �g/ml) and lysed. Expression ofphosphorylated RelA (S536), phosphorylated ERK1/2 (T202/Y204), SHP-2, and �-actin was analyzed by Western blotting. (D) qPCR of Cxcl1 and Cxcl5mRNAs after 2 h of treatment with LPS (12.5 �g/ml). Relative Cxcl1 and Cxcl5 expression was normalized with the housekeeping gene Tubb5. The dataare representative of three independent experiments. **, P � 0.01; ***, P � 0.001. The error bars indicate the SEM.

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might speculate that SHP-2 deficiency sensitizes IECs to the estab-lishment of microflora in the gut. In this context, pattern recog-nition receptor signaling in IECs contributes to the immune sur-veillance of enteric bacteria during early stages of hostcolonization (58). The beneficial effect of antibiotics observed inour model highlights the role of the microflora in the initiation ofinflammation in SHP-2IEC-KO mice. Both clinical observationsand animal studies supported the significance of intestinal micro-flora in the pathogenesis of IBDs (59).

During the neonatal period, the young mouse is exposedthrough mucosal surfaces to a plethora of environmental macro-molecules and microbial agents. Our data indicate that epithelialSHP-2 controls responses to commensal bacteria by limiting del-eterious proinflammatory immune responses in the lamina pro-pria. Thus, epithelial SHP-2 is a novel regulator of intestinal in-flammation. Importantly, we found that SHP-2 expression issignificantly reduced in patients with ulcerative colitis. SinceSHP-2 is expressed not only in IECs, but also at high levels inimmune cells and fibroblasts, further studies with microdissectedcolonic samples from inflamed epithelium, adjacent stroma, andnormal epithelium are needed to clearly determine if alterations inepithelial SHP-2 expression are a predisposing factor or are onlyassociated with the development of inflammation.

ACKNOWLEDGMENTS

We thank Anne Vézina, Marie-Josée Langlois, Evelyne Roy, and ÉricTremblay for technical assistance and Claude Asselin for critical readingof the manuscript.

Geneviève Coulombe is an NSERC Alexander Graham Bell studentscholar. Étienne Lemieux holds an FRSQ student scholarship. NathalieRivard, François Boudreau, and Nathalie Perreault are members of theFRSQ-funded Centre de Recherche Clinique Étienne-LeBel. FrancoisBoudreau and Nathalie Perreault are senior scholars from FRSQ. NathalieRivard holds a Canadian Research Chair in Colorectal Cancer and Inflam-matory Cell Signaling. This research was supported by the Crohn’s andColitis Foundation of Canada (N. Rivard).

We have no competing interests.

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