Microbiota matures colonic epithelium through a coordinated induction of cell cycle-related proteins in gnotobiotic rat

doi:10.1152/ajpgi.00384.2009 299:G348-G357, 2010. First published 13 May 2010;Am J Physiol Gastrointest Liver Physiol

Camille Mayeur, Pierre-Henri Duée, Philippe Langella and Muriel ThomasClaire Cherbuy, Edith Honvo-Houeto, Aurélia Bruneau, Chantal Bridonneau,gnotobiotic ratcoordinated induction of cell cycle-related proteins in Microbiota matures colonic epithelium through a

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Microbiota matures colonic epithelium through a coordinated induction of cellcycle-related proteins in gnotobiotic rat

Claire Cherbuy,1 Edith Honvo-Houeto,1 Aurélia Bruneau,1 Chantal Bridonneau,1 Camille Mayeur,1

Pierre-Henri Duée,2 Philippe Langella,1 and Muriel Thomas1

1Institut National de la Recherche Agronomique (INRA), MICALIS, UMR1319, Pôle Ecosystèmes, Jouy-en-Josas; and 2INRA,Versailles, France

Submitted 28 September 2009; accepted in final form 6 May 2010

Cherbuy C, Honvo-Houeto E, Bruneau A, Bridonneau C,Mayeur C, Duée PH, Langella P, Thomas M. Microbiota ma-tures colonic epithelium through a coordinated induction of cellcycle-related proteins in gnotobiotic rat. Am J Physiol GastrointestLiver Physiol 299: G348 –G357, 2010. First published May 13,2010; doi:10.1152/ajpgi.00384.2009.—Previous studies have sug-gested that intestinal microbiota modulates colonic epithelium re-newal. The objective of our work was to study the effects of micro-biota on colonic epithelium structure and cell cycle-related proteins byusing gnotobiotic rats. Colonic crypts and amount of cell cycle-relatedproteins were compared between germ-free (GF), conventional (CV),and conventionalized rats by histochemistry and Western blot. Ki67and proliferating cell nuclear antigen (PCNA) were used as surrogatesfor proliferative cells; p21cip1 and p27kip1 were markers of cell cyclearrest; anti- and proapoptotic proteins, Bcl2 and Bax, respectively,were also studied. We observed 40% increase of the crypt prolifera-tive area 2 days after inoculation of GF rats with a complex micro-biota. This recruitment of proliferative cells may account for the 30%increase of crypt depth observed between CV and GF rats. Thehyperproliferative boost induced by microbiota was compensated by afourfold increase of p21cip1 and p27kip1 involved in cell cycle arrestand a 30% drop of antiapoptotic Bcl2 protein while Bax was un-changed. Inductions of p21cip1, p27kip1, and PCNA protein were notparalleled by an increase of the corresponding mRNA. We alsoshowed that p21cip1 induction by microbiota was partially restored byBacteroides thetaiotaomicron, Ruminococcus gnavus, and Clostrid-ium paraputrificum. Colonization of the colon by a complex micro-biota increases the crypt depth of colon epithelium. This event takesplace in conjunction with a multistep process: a hyperproliferativeboost accompanied by compensatory events as induction of p21cip1

and p27kip1 and decrease of Bcl2.

intestine; germ-free; PCNA; Bcl2; p21cip1; p27kip1

THE GASTROINTESTINAL TRACT of mammals is colonized by acommunity of microorganisms reaching levels as high as 1011

bacteria/g of content in the colon. The intestinal microbiotaincludes more than 1,000 different species of bacteria belong-ing mainly to two divisions, the Firmicutes and the Bacte-roidetes. Microbiota represents a large genomic repertoire asrevealed by metagenomic approaches (12) and an efficaciousorgan to recover energy (33). Intestinal microbiota has co-evolved with its host, exploiting mutualistic interactions (24).Thus the gut immune tolerance toward microbiota partly in-volves fine-tuned regulation of the transcription factor NF-�Bby commensal bacteria (19). Furthermore, microbiota induceexpression of fucosylated cell surface glycans by mammalian

gut epithelium (4, 29), which may be used as receptors forbacterial adhesins or as energy sources for the intestinal bac-teria, especially in limited nutrient availability (45). Finally,our group has shown that intestinal microbiota diversifiesmetabolic pathways of the colonic mucosa, especially the oneof butyrate, one of the end products of bacterial fermentation(5) and a major energy source for colonocytes. Hence, the crosstalk between these two obligate partners (i.e., microbiota anddigestive tract) leads to the development of a stable ecosystem(24, 42).

Colonic epithelium is one of the most dynamic structures inthe whole organism as it self-renews every several days. Thisrenewal is sustained by epithelial progenitor cells that migrateupward from the bottom of the crypts undergoing proliferation,differentiation, and maturation before their extrusion into theintestinal lumen (3, 28). It is now well established that somepathogenic bacteria, probably for their own benefit, can modifythe host cell cycle (35). This point has recently been revealedfor Shigella, a pathogenic intestinal bacteria, which stronglyreduces the number of proliferating cells in a rabbit ileal loop(16). However, this effect on cell cycle may not be producedexclusively by pathogenic strains (34).

Indeed, the commensal intestinal microbiota influences cellproliferation and cell kinetic variables of the intestinal epithe-lium in gnotobiotic animals. An increased duodenum epithe-lium proliferation (23) and enterocyte transit time from crypt tovillus (43) were observed in conventional (CV) mice comparedwith germ-free (GF) ones. More recent results indicate that theileal expression level of some genes involved in apoptotic andproliferative activities are modified in the presence of a com-plex microbiota (48). In the colon, the number of cells per cryptis reduced in GF compared with CV animals (1). Therefore,throughout the digestive tract, all indicators linked to cellproduction and cell turnover are slower in the absence ofmicrobiota.

To better understand how microbiota modulates the cellcycle in the digestive tract, the objectives of our work were tostudy 1) the effects of microbiota colonization on colonicepithelium structure and on cell cycle-related proteins by usinggnotobiotic rats and 2) whether the effect of complex micro-biota on cell cycle-related proteins may be mimicked bydominant members of the intestinal microbiota: Bacteroidesthetaiotaomicron, Ruminococcus gnavus, and Clostridiumparaputrificum. These strains belong to Bacteroidetes andFirmicutes phyla. Furthermore, effects of B. thetaiotaomicronon the host intestinal response indicate the major role of thisbacteria in symbiotic host-bacteria relationships (4, 19).

Address for reprint requests and other correspondence: C. Cherbuy, INRA,MICALIS (UMR1319), Pôle Ecosystèmes, Domaine de Vilvert, 78 352 Jouy-en-Josas Cedex, France (e-mail: [email protected]).

Am J Physiol Gastrointest Liver Physiol 299: G348–G357, 2010.First published May 13, 2010; doi:10.1152/ajpgi.00384.2009.

0193-1857/10 Copyright © 2010 the American Physiological Society http://www.ajpgi.orgG348


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This work shows that, with along colonization, the micro-biota increases crypt depth in coordination with an induction ofcell cycle-related proteins.

MATERIALS AND METHODS

Animals and experimental design. All procedures were carried outaccording to European guidelines for the care and use of laboratoryanimals and with permission 78–122 of the French Veterinary Ser-vices. The following groups of rats (male, Fisher 344) were used:germ-free (GF); conventional (CV); GF inoculated with a fecalmicrobiota obtained from CV rats (Ino-CV); GF-inoculated with purebacterial strains: C. paraputrificum (strain number: 217.59) (30)isolated from the intestinal contents of CV rats (Ino-Cp); B. thetaio-taomicron (strain number: BtII8) (Ino-Bt) (11) and R. gnavus (strainnumber: FRE1) (Ino-Rg) (9), isolated from the fecal contents ofhumans.

To obtain Ino-CV rats, GF rats were inoculated by oral gavage with1 ml feces freshly recovered from a CV rat and diluted 100-fold inLCY (Liquid Casei Yeast extract) medium. To obtain monoxenic rats,GF rats were inoculated by oral gavage with 1 ml of an 18- to 24-hanaerobic culture.

Animals were born and bred at the Centre de Recherches, InstitutNational de la Recherche Agronomique (Jouy-en-Josas, France). TheGF, Ino-Cp, Ino-Bt, and Ino-Rg rats were reared in Trexler-typeisolators (La Calhène, Vélizy, France). The CV and Ino-CV batcheswere reared in standard conditions. All groups of rat received the samestandard diet (UAR), with the exception of a batch of Ino-Cp ratswhich received a specific diet containing both amylomaize starch andlactulose to stimulate butyrate production (5, 30). GF and CV ratscompared with this Ino-Cp group received the same diet. All dietswere sterilized by gamma irradiation (45 kGy). All rats were eutha-nized at the age of 3 mo. In the group of Ino-CV, rats were euthanized2, 14, or 30 days after the inoculation with conventional microbiota;they were named Ino-2d, Ino-14d, and Ino-30d, respectively. Monox-enic rats were euthanized 30 days after inoculation with a purebacterial strain.

At 9 AM, rats were anesthetized with isoflurane, the colons wereremoved and immediately used either for cell isolation or for histo-logical procedure.

Cell isolation procedure. Colonic epithelial cells were isolatedfrom the whole colon according to the method described by Cherbuyet al. (6). Cells originating from the whole epithelium were obtainedunder this procedure (6). The cell pellet was immediately used forprotein extraction.

Protein extraction. Protein extraction was made on freshly isolatedcells according to Ref. 22. Briefly, the cell pellet was resuspended ina lysis buffer (22) containing 0.1% Triton X-100 and a cocktail ofprotease inhibitor (Roche). Lysis was performed for 1 h on a contin-uous rotation at 4°C. During lysis, cells were homogenized twicethrough a 26-gauge needle. Cells were centrifugated (10,000 g; 4°C;20 mn), the supernatant was removed, aliquoted, and stored at �80°Cuntil analysis. Proteins were measured according to Ref. 25.

Western blot analysis. Proteins were resuspended in Laemmlisolution heated 5 min at 90°C and electrophoresis was run on a 12 or15% SDS-PAGE. After electrophoresis, proteins were transferredonto polyvinylidene difluoride membrane (Amersham Biosciences,Saclay, France). After blocking by TBS-T/5% milk, membranes wereincubated overnight at 4°C with the primary antibody, followed byincubation with appropriate peroxidase conjugated secondary antibod-ies (Jackson ImmunoResearch Laboratories, West Grove, PA). Thesignal was detected using the ECL � kit (Amersham Biosciences).Proteins were analyzed using anti-PCNA (GeneTex; diluted 1/1,000),anti-p21cip1 (Oncogene; 1/200 or Santa Cruz Biotechnology; 1/200),anti-p27kip1 (Santa Cruz Biotechnology; 1/200), anti-Bcl2 (SantaCruz; 1/400), anti-Bax (Santa Cruz; 1/200), anti-skp1 (BD Transduc-tion Laboratories; 1/1,000), anti-GAPDH (assay designs, 1/5,000).

Signals detected on autoradiographic films were quantified by scan-ning densitometry of the autoradiograph by using a Lasplus camera(Fujifilm, Paris, France) and Aida software (Raytest, Courbevoie,France).

Histology and immunohistochemistry. Colon samples were cut into2-cm sections, fixed in 4% paraformaldehyde (5 h, room temperature),dehydrated, and embedded in paraffin according to standard histolog-ical protocols. Four-micrometer sections were mounted on SuperFrostPlus slides. Slides were stained with eosin for histological analysis.Immunological staining was done with the Envision� system-horse-radish peroxidase (Dako, France) according to the recommendationsof the manufacturer. Antigen retrieval was performed by boiling slidesfor 40 min in 0.01 mol/l sodium citrate pH 6.0. Primary antibodiesused were Ki67 (clone MIB-5, Dakocytomation, dilution 1/50) andanti-PCNA (GeneTex; dilution 1/10,000). Negative controls wereperformed by omitting the primary antibody from the reactions. Inthese conditions no signal was observed (data not shown). OnlyU-shaped longitudinally cut crypts with open lumina along the cryptaxis were evaluated. For each section, ki67- or PCNA-positive cellswere counted on 10 crypts, and results were expressed as percent oftotal colonic crypt cells.

Total RNA extraction and real-time RT-PCR analysis. Total RNAwas extracted from isolated colonic epithelial cells by the guani-dinium thiocyanate method (7). RNA concentration and purity weredetermined by absorbance measurement using a nanodrop and RNAIntegrity Number (RIN) was checked with the Agilent 2100 bioana-lyzer and the RNA 6000 nano labChip kit (Agilent technologies). AllRNAs had a RIN between 8.5 to 9.5, indicating a high RNA qualityin all samples. Seven micrograms of the total RNA samples weresubjected to a reverse transcription step by use of the high-capacitycDNA archive kit (Applied Biosystems, France). Real-time quantita-tive PCR was performed on cDNAs in a ABI PRISM 7000 SequenceDetection System by using Rn00582195-m1 and Rn00589996-m1TaqMan Gene expression assays for p21cip1 and p27kip1, respectively(Applied Biosystems, France). Since we have already used 18S rRNAwith colon samples (18), 18S rRNA was considered as housekeepinggene and measured with Hs99999901-s1 Taqman gene expressionassays. Results obtained on p21cip1 and p27kip1 were normalized to18S rRNA (reference gene) and compared with the means target geneexpression of CV rats as calibrator sample. At least three rats wereused for each GF, Ino-2d, Ino-14d, Ino-30d, and CV group. Thefollowing formula was used: fold change � 2��Ct, where ��threshold cycle (Ct) equals (target Ct � reference Ct) of sample minus(target Ct � reference Ct) of the calibrator.

Presentation and analysis of data. Results are presented as means �SE for the number of animals indicated. Comparisons of group datawere performed using one-way analysis of variance (ANOVA) fol-lowed by a Tukey’s Studentized range test (jmp version 7) when theANOVA revealed differences among the groups. Differences wereconsidered statistically significant at P � 0.05.

RESULTS

The microbiota shapes the morphology of the colonicepithelium. Colonic crypt depth was greatly affected by thebacterial status of the rats with a crypt depth 35% lower ingerm-free rats (208 � 32 �m for GF rat vs. 274 � 19 �m forCV rats; P � 0.05) (Fig. 1A). Furthermore, in the colonicmucosa of GF rats, bifurcating crypts were observed with twoflask-shaped bases joining in a single unit at the top of the crypt(Fig. 1A). These features are characteristic of crypt fission,which is a mechanism whereby intestinal crypts divide (10).Crypts with a fission form represented 25.8 � 18.6% of totalcrypt in GF rat mucosa, which was significantly higher thanvalues obtained in CV (2.7 � 3.9% of total crypt).

G349MICROBIOTA AND COLONIC CELL-CYCLE RELATED PROTEINS

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The microbiota modulates cell cycle-related protein abundance. Itis well known that colonic crypts are highly spatially structuredwith an area where the cells are proliferating and a nonprolif-erating portion at the top (3, 28). As expected, the proliferativecells, which were stained by two proliferative markers (Ki67and PCNA) (Fig. 1, B and C), were restrained to the bottom ofthe crypt. The Ki67-positive cells represented, respectively, 41 � 5vs. 46 � 4% of total colonic crypt cells in GF and CV rats. ThePCNA-stained cells were 63 � 5% in GF and 65 � 5% of totalcells in CV rats. There were thus no significant differences inthe distribution of Ki67 and PCNA in CV and in GF rats. As

it has been previously reported (15, 32), the PCNA-stainedzone was more extended than the Ki67-positive zone.

The amount of PCNA was compared by Western blot incolonic epithelial cells from GF and CV rats (Fig. 2). ColonicPCNA protein levels were 60% higher in CV than in GF rats(Fig. 2A). It has recently been shown that Cif, a cyclomodulinproduced by pathogenic strains of Escherichia coli, modulatesthe amount of two cyclin-dependent kinase inhibitors, p21cip1

and p27kip1 (41). According to the key role of these twoproteins in intestinal epithelial cell renewal (40), we furtherstudied the effect of the GF state on p21cip1 and p27kip. Results

Fig. 1. Histological analysis of the colonicepithelium of germ-free (GF) and conven-tional (CV) rats. GF and CV rats were eu-thanized at the age of 3 mo. A: representativephotomicrograph of colonic sections ob-tained from GF and CV rats stained witheosin. B and C: representative photomicro-graph of immunohistochemical staining withKi67 (B) and with PCNA (C) in the midco-lon of GF and CV rats. Graphs at rightrepresent measurements of crypt depth in�m (A), Ki67-positive cells (B), and PCNA-positive cells (C) expressed as a percentageof total colonic crypt cells. Results are pre-sented as means � SE for n � 7 rats pergroup. *Statistically significant differences(P � 0.05) between groups. Arrows in theeosin-stained section (A) indicate crypt fis-sion in GF rat colonic epithelium. Magnitudeused for photomicrographs: 20.

G350 MICROBIOTA AND COLONIC CELL-CYCLE RELATED PROTEINS



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indicated that abundance of these proteins was dramaticallydecreased in GF rat (see Fig. 2B for p21cip1 and Fig. 2C forp27kip1). All these quantitative data are in accordance with thefact that CV rats displayed deeper crypts with a well-preservedratio between proliferative and nonproliferative cells as ob-served by immunohistochemistry (Fig. 1, B and C).

Progressive adaptation of colon epithelium to the presenceof microbiota. A progressive effect of the transfer of a complexintestinal microbiota on the colonic epithelium was furtherstudied. Ino-CV rats were euthanized 2, 14, or 30 days afterinoculation. Several arguments were considered for the choiceof this time course: we chose a time point preceding the totalrenewal of the colonic epithelium (2 days) and later points,encompassing two or three renewals of colonic epithelium (14and 30 days) (3, 28). These periods also correspond to theprogressive establishment of the biochemical functions of thetransferred intestinal microbiota, which occurs within 3 wkfollowing the transfer of the intestinal microbiota (31).

Histological analysis indicated that crypt depth progres-sively increased following the transfer of a complex microbiota

(Fig. 3A). The crypt depth was of 208 � 32 �m in GF rats, 234 �27 �m in Ino-2d, 274 � 27 �m in Ino-14d, and 256 � 4 �m inIno-30d. The maximal depth of crypt was reached in Ino-14d,since there were no significant differences between values ob-tained in Ino-14d and CV rats. Colonic crypt structure alsodiffered strongly between GF and Ino-CV. As previously men-tioned, typical figures of crypt fission were observed in the colonicmucosa of GF rats. The number of bifurcating crypts was de-creased in the colonic mucosa of Ino-2d, representing 8.33 �7.14% of total crypt. In Ino-14d or Ino-30d, we were not able toobserve such bifurcating crypts. Furthermore, in contrast to GF,colonic crypts were tightly and individually structured in Ino-CVgroups. According to these observations, one can postulate that inGF rats colonic crypts are ready to split up, which can account forthe higher number of crypts in Ino-2d. Indeed, in this group ofrats, the crypt number was significantly 30% higher than in GFrats. Thus the number of crypts per millimeter of colonic lengthwas respectively of 20.0 � 3.0 and of 26.6 � 3.5 for GF andIno-2d groups (P � 0.05). In Ino14d and Ino-30d the crypt densitywas progressively decreased respectively to 22.8 � 2 and to

Fig. 2. Western blot analysis of PCNA,p21cip1, and p27kip1 in colonic epithelialcells of GF and CV rats. RepresentativeWestern blot autoradiography obtained withtotal proteins of colonic epithelial cells iso-lated from GF and CV rats. Total proteins, 5�g or 30 �g, respectively, for PCNA (A) andfor p21cip1 (B) and p27kip1 (C) analysis, weresize fractionated and blotted with adequateprimary antibodies. SKP1 and gapdh pro-teins were used as internal controls. Densi-tometric analysis of autoradiography per-formed on n � 10 rats per group. Results arepresented as means � SE. *Statistically sig-nificant differences (P � 0.05) betweengroups.




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20.9 � 2.5 crypts/mm. This was not different from CV values(21.2 � 1.5 crypts/mm).

When probing Ki67- and PCNA-positive cells (Fig. 3, B andC), the presence of complex microbiota for 2 days led to a

rapid increase in the percent of stained cells. As previouslymentioned, Ki67-positive cells represented 41 � 5% of thetotal colonic crypt cells in GF, whereas they represented up to73 � 5% in Ino-2d. PCNA-positive cells ranged from 63 � 5%

Fig. 3. Modification of colonic mucosa structure after the transfer of a CV fecal microbiota. GF rats were inoculated with a fecal microbiota coming from thefeces of CV rats. Conventionalized rats were then euthanized 2, 14, or 30 days after the inoculation (Ino-2d, Ino14d, Ino-30d) at 3 mo. A: representativephotomicrograph of colonic sections stained with eosin obtained from GF, Ino-2d, Ino-14d, and Ino-30d rats. B and C: representative photomicrograph ofimmunohistochemical staining Ki67 (B) and PCNA (C) in the midcolon of GF, Ino-2d, Ino-14d, and Ino-30d rats. Graphs at right represent measurements ofcrypt depth in �m (A), Ki67-positive cells (B) and PCNA-positive cells (C) expressed as a percentage of total colonic crypt cells. Results are presented as means �SE for n � 7 to 5 rat per group. Means with different letters are significantly different (P � 0.05). Magnitude used for photomicrographs: 20.




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in GF to 88 � 4% in Ino-2d rats. When microbiota was presentfor a longer time, i.e., 14 and 30 days, neither Ki67- norPCNA-stained cells were significantly different from that inCV rats. These results indicated that the inoculation of acomplex fecal microbiota in GF rats led to a transitory hyper-proliferation boost that was detected at 2 days after inoculation.

As observed by immunohistochemistry, we confirmed byWestern blot that PCNA abundance was higher in Ino-2d

compared with GF, thus acting as a molecular signature of theboost in cell proliferation (Fig. 4A). The first days of bacterialestablishment was also accompanied by a 30% decrease ofBcl2, an antiapoptotic regulator, but had no effect on theproapoptotic protein Bax (Fig. 4B). In contrast to PCNA andBcl2, neither p21cip1 (Fig. 4C) nor p27kip1 (Fig. 4D) wasmodified between GF and Ino-2d rats. However, both proteinswere increased in Ino-14d, i.e., 14 days after inoculation at the

Fig. 4. Evolution of PCNA, Bcl2, Bax, p21cip1, and p27kip1 protein amount in colonic epithelial cells after the transfer of a CV fecal microbiota. GF rats wereinoculated with a fecal microbiota coming from CV rats. Ex-GF rats were then euthanized 2, 14, or 30 days after the inoculation (Ino-2d, Ino-14d, Ino-30d).Proteins were extracted from isolated colonic epithelial cells as described in MATERIALS AND METHODS and analyzed by Western blot. Representativeautoradiographs respectively obtained against PCNA (A), Bcl2/Bax (B), p21cip1 (C), and p27kip1 (D). SKP1 was used as an internal loading control. Graphs atright: densitometry analysis of autoradiography for PCNA, Bcl2/Bax, p21cip1, and p27kip1, performed on n � 7–10 rats per group. Results are presented as means �SE. Means with different letters are significantly different (P � 0.05).




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same level as in CV rats (Fig. 4, C and D). The p21cip1 andp27kip1 values obtained for Ino-14d were similar to Ino-30d(see graphs of figures). We also confirmed this inductionpattern by using nuclear proteins for PCNA, p21cip1, andp27kip1 (see Supplementary Fig. S1; Supplemental Material forthis article is available online at the Journal website).

Thus quantitative data obtained by Western blot suggestedthat the inoculation of a complex fecal microbiota in GF ratsled to a hyperproliferation step 2 days after inoculation and asuccessive induction of cell cycle arrest proteins. This was alsoaccompanied by a decrease in the antiapoptotic protein Bcl2whereas the proapoptotic protein Bax amount remained un-changed. Also, we did not observed cleaved forms of caspase8 and caspase 3 nor increase in these protein amounts amonggroup of rat (data not shown).

In contrast to protein levels, mRNA encoding p21cip1,p27kip1, and PCNA were not modified by the bacterial status.Indeed, 2��Ct values for p21cip1 were 1 � 0.3, 0.94 � 0.2, 0.6 �0.12, and 1.2 � 0.4, respectively, from GF, Ino-2d, Ino-14d,and CV rats. For p27kip1, 2��Ct values were 1.8 � 0.5, 1 �0.3, 0.7 � 0.1, 1 � 0.1, respectively, from GF, Ino-2d,Ino-14d, and CV rats. Similar mRNA amounts for p21cip1 andPCNA were also observed by Northern blot in GF and CV rats(data not shown). In parallel, we also observed that p53 isstable in colonocytes whatever the bacterial status of the rat(data not shown).

The effect of a complex microbiota is partially restored bydominant members of the adult intestinal microbiota. To definewhether the effects of a complex microbiota may be mimickedby B. thetaiotaomicron, R. gnavus, and C. paraputrificum, weobtained monoassociated rats (Ino-Bt, Ino-Rg, Ino-Cp). Oneweek after the inoculation, bacteria were present in the feces at109 to 1010 bacteria/g and remained at a high level throughoutthe protocol (30 days) (see Supplementary Fig. S2 and Refs. 5,30). Cell cycle proteins were analyzed on colonic epithelialcells by Western blot (Fig. 5; see Supplementary Fig. S3 forrepresentative autoradiographs). None of the three bacterialstrains modulated PCNA protein abundance (Fig. 5, A–C).Also, p27kip1 amount was unchanged between GF, Ino-Bt andIno-Rg (data not shown). In contrast, p21cip1 increased weaklyin monoxenic rats, while remaining lower than the amountdetected in CV rats (Fig. 5, D–F).

It has been previously shown that C. paraputrificum have theability to produce butyrate when rats are fed a diet containingboth amylomaize starch and lactulose (5, 30). The levels ofPCNA (Fig. 5C) and p21cip1 (Fig. 5F) were unchanged be-tween Ino-Cp rats fed either a standard (UAR diet) or anamylomaize starch- and lactulose-enriched diet (EL5 diet).

DISCUSSION

This work shows for the first time that microbiota modifiesin vivo colonic epithelium structure in concordance with awell-orchestrated induction of proteins involved in the cellcycle. Results indicate that colonization of the colon by acomplex microbiota increases the crypt depth of colonic epi-thelium that is related to a hyperproliferative boost and com-pensatory events illustrated by the drop of Bcl2 and theinduction of p21cip1 and p27kip1. These events led to a con-trolled homeostasis of the colonic epithelium in response to thepresence of a complex intestinal microbiota. We have recently

also observed a controlled hyperplasia of the colonic epithe-lium in human patients with short bowel syndrome, displayinga drastic change of microbiota composition (17, 18). Thepreservation of homeostatic balance is also crucial in protect-ing intestine from injury such as chemical challenges or radi-ation (39). Since Toll-like receptors are involved in bacterialrecognition and play an important role in intestinal epithelialhomeostasis control (38), it would be now interesting to followup these receptors in GF and different groups of inoculatedrats.

Overall, all observations led us to conclude that the absenceof microbiota is associated with an epithelium atrophy. Bycomparing GF, CV, and Ino-CV rats, we determined that ratsharboring a complex microbiota had deeper colonic crypts(Fig. 1 and 3). Furthermore, we observed a lower number ofcolonic crypts in the mucosa of GF rats than in Ino-2d rats,suggesting that the implantation of microbiota contributes tocrypt formation, probably through crypt fission. This resultmust be put in perspective with the colonic maturation throughcrypt fission that occurs after birth in mammals (27). The factthat microbiota is a key actor in recovering energy from foodis obviously linked to the metabolic activity of microorganismsbut may also be related to its trophic effect on the epitheliumsurface. The increase in the intestinal absorptive surface areacould thus contribute to the weight gain observed in CVcompared with GF rodents (2).

Our observations, in accordance with previous data (1),confirm that colonic epithelial cells of GF had a lower capacityof cycling than CV cells. The fact that GF displayed higheramount of antiapoptotic protein like Bcl2 than CV rats indicatethat all life cycle steps of epithelial cells (from division toapoptosis via cell cycle arrest) can be modulated by micro-biota. The effect of the commensal microbiota on cell cycleproteins could explain the acquisition of a deeper colonicepithelium structure with a well-preserved ratio between pro-liferating and nonproliferating compartments. In addition, ithas been shown that the rate of intestinal renewal provides animportant intrinsic defense system. The intestinal cell turnoverrate is one of the key factors involved in resistance towardparasitic infection, probably because it participates in its ex-pulsion (8). The trophic effect of microbiota could thus also tobe a manner for the host to stimulate the mucosal defense.Throughout the digestive tract, the effect of microbiota on cellcycle related proteins was segment specific and particularlymarked in the colon. In the ileum, PCNA, p21cip1 and p27kip1

were only 30–40% higher in CV than in GF rats (vs. 60/70%in colon), whereas in duodenum and in jejunum these proteinstended to be similar in CV and GF rats (data not shown). Thusthe colon where the density of microbiota was the highestseems to be the site where the cell cycle related proteins weremore sensitive to microbiota.

PCNA and Ki67 are abundant proteins in the colon withrestricted detection at the bottom of the crypts. Correlated withtheir localization, Ki67 and PCNA play a role in proliferation(15, 20, 32): Ki67, although universally used as a proliferativemarker, still has an unknown function; PCNA is a member ofthe DNA sliding clamp family and plays a role not only inproliferation through DNA replication but also in DNA repair(26). The fact that both Ki67 and PCNA were enhanced 2 daysafter microbiota transfer illustrated a rapid and strong boost inproliferation. Because the increase in the proliferative compart-




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ment precedes complete renewal of the colonic epithelium, ourresults suggest that microbiota signals may mobilize stem cellsto generate more proliferative cells. When the pattern ofprotein expression (Fig. 4) was paralleled with the modeling ofthe epithelium structure (Fig. 3), the proproliferative event wasanterior to the maximal depth acquisition of the crypt (ob-served at 14 days after inoculation). Hence, on the basis of thiskinetic, we propose that the proproliferative bacterial signal isnecessary to provide deeper crypts. The boost of proliferationwas also accompanied by a decreased of Bcl2 protein amountthat regulates the number of cells entering the bottom/top axisin the colon (47). All these observations suggested that a tightcontrol of epithelium homeostasis began as soon as 2 days aftermicrobiota inoculation activating proproliferative and decreas-ing antiapoptotic signals. Furthermore, in addition to its role inthe proliferative process, PCNA could be of physiological

importance for protecting epithelial cells toward DNA dam-ages linked to bacteria products. Indeed, it has been demon-strated that the action mechanisms of several cyclomodulinsinvolve eukaryotic DNA damage (34, 35).

P21cip1 and p27kip1 are cyclin-dependent kinase inhibitorsinvolved in cell cycle arrest, and they are considered tumor-suppressor genes. In the intestine, their role is probably morecomplex (37, 44). As an example, there are no differencesbetween proliferation and differentiated epithelial cell lineagesin p21cip1�/�, p27kip1�/�, and p21cip1�/�,p27kip1�/� micecompared with their wild-type counterparts (50). It has there-fore been suggested that p21cip1 and p27kip1 play a critical rolein regulating intestinal cell turnover after external stress. In-deed, disruption of these genes enhanced intestinal tumorformation after administration of a carcinogen (36) or a high-fat “Western style” diet (49). According to these data, our

Fig. 5. Analysis of PCNA and p21cip1 in co-lonic epithelial cells isolated from monoasso-ciated rats. GF rats were inoculated with Ru-minococcus gnavus (Ino-Rg) (A and D), Bac-teroides thetaiotaomicron (Ino-Bt) (B and E),or Clostridium paraputrificum (Ino-Cp) (C andF) and PCNA (A–C) and p21cip1 (D–F) wereanalyzed on colonic isolated cells. All ratswere euthanized 30 days after the inoculationwith 1 ml of overnight bacterial culture. Ino-Rg, Ino-Bt, and a group of Ino-Cp were fed astandard diet (UAR). To promote butyrate pro-duction in Ino-Cp, another group of Ino-Cpwas fed the EL5 diet. Colonic epithelial cellswere isolated and proteins were extracted asdescribed in MATERIALS AND METHODS. Pro-teins were size fractionated and blotted with ananti-PCNA and an anti-p21cip1 (see Supple-mentary Fig. S3 for representative autoradio-graphs). Membranes were then analyzed fordensitometry analysis performed on n � 5–4rats per group. Results are presented as means� SE. Means with different letters are signif-icantly different (P � 0.05).




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results suggest that the colonic hyperplasia observed within 2days after transfer of intestinal microbiota is counterbalancedby an induction of p21cip1 and p27kip1, thus restraining prolif-eration. Induction of p21cip1 and p27kip1, which are bothinvolved in gating cell-cycle progression, may attenuate themicrobiota-induced proproliferative signals in the intestine.

We sought to use monoassociated rats to decipher mecha-nisms involved in the effect of complex microbiota on cellcycle-related proteins. Dominant members of the adult intesti-nal microbiota, i.e., B. thetaiotaomicron and R. gnavus, werechallenged for their ability to mimic a complex microbiota.Furthermore, we tested the role of butyrate through C. para-putrificum-inoculated rats receiving two diets, producing bu-tyrate or not. None of these conditions increased PCNA proteinlevels, although each pure bacterial strain had been inoculatedat the same level as the complex microbiota (fecal dilution wasof 1/100 leading to 109 bacteria/ml fecal bacteria). The hyper-proliferative boost thus seems to be related more to the diver-sity of bacterial population than to the amount of bacteriapresent in the colon. It has been shown that when a complexfecal microbiota was transferred into GF rodents it evolved inthe first days following transfer (14). After inoculation, aerobicbacteria (as Enterobacteriaceae, Streptococcus) predominate,whereas anaerobic bacteria (as Eubacterium) are in a minority.At 2 or 3 days after the transfer, these proportions begin to beinverted and tend to be closer to the “classical” adult compo-sition of the microbiota. Thus modifications that we observedon cell cycle-related proteins in the presence of a complexmicrobiota can be explained by a sequence of events occurringin healthy colonic mucosa toward some environmental stimulibut also by the progressive evolution of the bacterial speciescomposing the intestinal content after inoculation. The role ofthe pioneer bacteria toward the hyperproliferative boost shouldbe studied. It is noteworthy that B. thetaiotaomicron and R.gnavus, which are obligate anaerobes, did not modify PCNAnor p27kip1 amounts but, in contrast, acted on the late inductionof p21cip1. According to results obtained with C. paraputrifi-cum-inoculated rat, butyrate did not seem to be involved in thisinduction. In addition, mRNA content of p21cip1 was similarbetween GF and CV, thus reinforcing our hypothesis thatbutyrate (a transcriptional activator of p21cip1) was not in-volved in p21cip1 microbiota-related activation. Being giventhat P21cip1 and p27kip1 mRNAs remained stable between GFand CV rats and that many microbes interfere with differentstage of posttranslational pathways in cell cycle progressionprocess (13, 46), we are currently studying whether commensalpopulation could stabilize these proteins maybe through theproteasome machinery (21).

Up to now, p21cip1 and p27kip1 were known to be targeted bytoxins produced by pathogenic bacteria. Here, we have dem-onstrated that intestinal microbiota can also modulate similarcell cycle-related pathways as pathogenic bacteria. Althoughnumerous studies concern the cross talk stalled between mi-crobiota and the host immune system, we have here shown thatmicrobiota is a major actor in modeling the epithelium struc-ture and in modulating eukaryotic cell cycle-related proteins.

ACKNOWLEDGMENTS

We thank Rosa Durao and Marie-Louise Noordine (MICALIS, INRA,Jouy-en-Josas, France) for technical help.

GRANTS

This work was supported by Syndifrais (42, rue de Châteaudun-75314PARIS Cedex 09) and by INRA (support of a young team).

DISCLOSURES

No conflicts of interest are declared by the author(s).

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Microbiota matures colonic epithelium through a coordinated induction of cell cycle-related proteins in gnotobiotic rat