British Journal of Nutrition

Transcription

1 (211), 16, q The Authors 211 doi:1.117/s x Fetl lipopolyscchride exposure modultes diet-dependent gut mturtion nd sensitivity to necrotising enterocolitis in pre-term pigs Mlene S. Cilieorg 1,2, Mette Schmidt 3, Kerstin Skovgrd 2, Mette Boye 2, Nicoli R. Weer 3, Peter M. Heegrd 2, Dougls G. Burrin 4 nd Per T. Sngild 1 * 1 Deprtment of Humn Nutrition, Fculty of Life Science, University of Copenhgen, 3 Rolighedsvej, DK-1958 Frederikserg C, Denmrk 2 Division of Veterinry Dignostics nd Reserch, Ntionl Veterinry Institute, Technicl University of Denmrk, DK-179 Copenhgen, Denmrk 3 Deprtment of Lrge Animls, Fculty of Life Science, University of Copenhgen, 3 Rolighedsvej, DK-187 Frederikserg C, Denmrk 4 USDA Children s Nutrition Reserch, Bylor College of Medicine, Houston, TX, USA (Received 1 July 21 Revised 18 Decemer 21 Accepted 2 Jnury 211 First pulished online 17 My 211) Astrct Uterine infections during pregnncy predispose to pre-term irth nd postntl moridity, ut it is unknown how prentl cteril exposure ffects mturtion of the immture gut. We hypothesised tht prentl exposure to grm-negtive lipopolyscchride (LPS) hs immunomodultory effects tht improve resistnce towrds necrotising enterocolitis (NEC) in pre-term neontes. At pproximtely 85 % gesttion, pig fetuses were injected intrmusculrly with sline or LPS ( 14 mg/kg), or intr-mnioticlly with LPS ( 4 mg/kg). Pigs were delivered y cesren section 3 5 d lter nd fed colostrum (C) or formul (F) for 48 h. Gut indices did not differ etween pigs injected intrmusculrly with sline or LPS, nd these groups were therefore pooled into two control groups ccording to diet (control-f, n 32 nd control-c, n 11). Control-F pigs showed reduced villus heights, mucosl structure, gut integrity, digestive enzymes, elevted NEC incidence (38 v. %, P, 5) nd severl differentilly expressed immune-relted genes, reltive to control-c pigs. Compred with the control-f nd control-c groups, vlues in formul-fed pigs given intr-mniotic LPS formul (n 17) were intermedite for villus height, enzyme ctivities, intestinl permeility nd NEC incidence (18 %, P¼ 2 reltive to control-f), nd numers of differentilly expressed immune genes. In conclusion, prentl exposure of the fetl gut to Grm-negtive cteri my modulte the immedite postntl response to n enterl diet nd colonising cteri. Key words: Fetl surgery: Premture irth: Intestinl inflmmtion Uterine infection during pregnncy predisposes to pre-term irth nd my e ssocited with n incresed overll neontl moridity. It remins uncler, however, how prentl cteril stimultion (systemic or luminl) ffects the sensitivity to inflmmtory responses nd necrotising enterocolitis (NEC) in the intestine of pre-term neontes (1 4). The immture intestine my show n incresed expression of the toll-like receptor (TLR)-4 to Grm-negtive lipopolyscchride (LPS), leding to n exggerted pro-inflmmtory response vi the NF-kB pthwy (5,6). In the immedite postntl period of term neontes, this innte immune response to LPS is down-regulted, fcilitting mucosl tolernce towrds Grm-negtive cteri nd generl cteril colonistion (5,6). Insufficient developmentl down-regultion of the TLR-4- medited response to Grm-negtive cteril stimuli my t lest in prt explin the exggerted inflmmtory response leding to NEC in pre-term neontes. An erly exposure to cteril stimuli my hve the potentil to enhnce epithelil immune development, s suggested y improved postntl lung mturtion nd the reduced respirtory distress syndrome following uterine infection in humn pregnncy (7 9). Correspondingly, intr-mniotic LPS (LPS) or IL-1 injection of fetl lms, mimicking uterine infection, is ssocited with n incresed numer of inflmmtory cells in the mniotic nd lung fluid, incresed surfctnt production nd incresed complince nd lung gs Arevitions: C, colostrum; control-c, control-colostrum; F, formul; control-f, control-formul; LPS, lipopolyscchride; LPS, intr-mniotic lipopolyscchride; LPS-F, intr-mniotic lipopolyscchride formul; NEC, necrotising enterocolitis; TLR, toll-like receptor. * Corresponding uthor: P. T. Sngild, fx þ , emil ps@life.ku.dk

2 Neontl gut response to fetl cteri 853 volume (1 12). The effect ws result of direct epithelil stimultion when the fetus inhled nd swllowed the mniotic fluid with cteril nd inflmmtory compounds (11). A corresponding epithelil stimultion nd incresed structurl, functionl nd immunologicl development could occur in the gstrointestinl trct nd ffect the NEC susceptiility of the immture intestine. A systemic stimultion with reltively high doses of LPS ws ssocited with cidosis nd deth in fetl lms (11) nd with NEC in pigs nd rts (13,14), indicting tht the route of cteril exposure (luminl or systemic) my influence the postntl response. Such proinflmmtory responses my e modified y diet just fter irth s shown in pre-term pigs, where the mother s colostrum with its high concentrtions of trophic nd immunomodultory compounds stimultes gut function nd suppresses pro-inflmmtory meditors nd NEC development reltive to formul (15 18). A pre-term neonte sujected to prentl systemic infection my thus enefit from colostrum feeding to counterct the pro-inflmmtory cteril insults. Given this ckground, the present study ws designed to test whether LPS dministrtion to fetl piglets ffects gut development nd immunologicl responses just fter irth, nd how different diets (formul or colostrum) my influence this response. Bsed on the effects reported previously for neontl lung function in lms, we hypothesised tht prentl LPS stimultion of fetl pigs would improve gut development, modulte the pro-inflmmtory responses nd improve cteril tolernce, nd therey reduce the diet-dependent NEC sensitivity of the immture intestine. Conversely, we hypothesised tht intrmusculr LPS injections might increse Fetl surgery nd injection 3 5 d Cesren delivery NEC sensitivity. In the present initil study, we tested reltively low doses of intrmusculr nd LPS, considering the high risk of premture lour nd fetl deth following intr-fetl surgery nd LPS dministrtion in lte gesttion. Structurl, functionl nd immunologicl indices of the gut tissue were mesured to identify the effects of intr-fetl LPS dministrtion s well s of the postntl diet. Methods nd mterils Prentl surgery nd lipopolyscchride injections All procedures in the niml protocol mentioned elow were pproved y the Ntionl Committee on Animl Experimenttion, Denmrk. At % gesttion, five pregnnt sows (Lrge White Dnish Lndrce) underwent lprtomy fter sedtion (zperone), nesthesi (propofol nd isoflurne fter intution) nd systemic (fentnyl) nd locl (lidocine) nlgesi tretment. The uterus ws exposed, nd in totl seventy-two live fetuses were injected with sline or LPS from Escherichi coli serotype K-235 (Sigm-Aldrich, St Louis, MO, USA) through the uterine wll (Fig. 1). Fetuses (estimted ody weights 1 kg) were injected intrmusculrly in the hind leg with either 5 ml sline (n 27) or 5 ml lowdose LPS ( 14 mg; n 23), or they were injected into the mniotic fluid vi the orl cvity with 1 ml high-dose LPS (totl 4 mg, n 22). Ech pig ws chipped for postntl recognition, nd the uterus nd fetuses were put ck into the dominl cvity nd the flnk ws closed y sutures. The sows were returned to the stles nd treted dily with rod-spectrum ntiiotics (moxycillin intrmusculrly) nd 2d Postntl feeding nd tissue collection (A) (B) (C) Intr-musculr LPS (n 23) Intr-musculr sline (n 27) Intr-mniotic LPS (n 22) Colostrum (n 5) Colostrum (n 6) Formul (n 14) Formul (n 18) Formul (n 17) Control-C (n 11) Control-F (n 32) LPS-F (n 17) Stillorn (n 11) Perintl deth (n 1) Fig. 1. Schemtic overview of the fetl interventions nd the groups of postntl pre-term pigs. Pig fetuses received sline or lipopolyscchride (LPS) injections (intr-mniotic or intrmusculr, (A)) 3 5 d efore pre-term delivery y cesren section (B) nd fed colostrum (C) or formul (F) for 2 d postntlly (C). For the tissue nlyses, dt from the intrmusculr sline nd LPS groups were pooled ccording to the diet (control-c nd control-f) nd compred with formul-fed pigs receiving intr-mniotic LPS (LPS-F).

3 854 M. S. Cilieorg et l. progesterone (5 mg) to void infections nd spontneous irth efore cesren delivery. Cesren section nd postntl feeding protocol Cesren section ws plnned to tke plce no lter thn 7 d fter the fetl surgery, unless signs of pre-term lour developed eforehnd, leding to sedtion, nesthesi nd immedite cesren delivery s descried erlier (15,16). A totl of sixty-one pigs were delivered nd immeditely trnsferred to temperture- nd oxygen-regulted incutors (Air-Shields, Htoro, PA, USA). Of the sixty-one pigs, one died soon fter irth from complictions not relted to NEC, leving sixty pigs to e included in the finl nlyses. Blood smples were drwn from the umilicl cord t cesren section nd t 6 h fter irth, nd from the hert t euthnsi to mesure lood gses, nd plsm hptogloin nd cortisol, s descried previously (15,16). Following our stndrd rering nd feeding protocol (15,16), the pigs were fitted with orogstric ctheters (6F; Phrmplst, Roskilde, Denmrk) nd fed full enterl nutrition (15 ml/kg per 3 h) for 2 d until euthnsi (pentoritone: 6 mg/kg, intrvenously) nd tissue collection. Pigs injected intrmusculrly were fed either cows colostrum (n 6 for sline nd n 5 for LPS injection, respectively) or infnt formul (n 18 for sline nd n 14 for LPS injection, respectively) (Fig. 1). All pigs given LPS were fed formul fter irth (n 17). The formul ws mde of three commercil products (per litre of wter: 8 g Pepdite 2-, 7 g Mxipro nd 75 ml Liquigen- MCT, kindly donted y SHS Interntionl, Liverpool, Merseyside, UK). The mcronutrient content ws designed to mtch the composition of sows milk nd contined per litre of formul: energy, 414 kj; protein (minly whey protein), 64 g; crohydrte (minly glucose), 45 g; ft, 61 g (sturted, 44 g; monounsturted 1 g; polyunsturted, 4 g); N, 3 g; K, 64 g; C, 59 g; P, 42 g (15). Bovine colostrum ws collected s the first milking fter prturition from diry cows (Holstein Friesin, Gjordslev Gods, Denmrk) nd diluted with 3 % wter to chieve n energy content similr to tht in the formul. Clinicl evlution nd tissue collection Pigs were checked every 3 h for NEC symptoms (loody dirrhoe, lethrgy, respirtory distress nd dominl distension). When these symptoms were evident, pigs were euthnised nd tissue ws collected. All remining pigs were euthnised on dy 3 (41 57 h postprtum). We evluted five gstrointestinl regions (stomch, proximl, mid nd distl smll intestine nd colon) for NEC lesions nd scored 1 6 ccording to our stndrd mcroscopic NEC pthology evlution: (1) sence of lesions; (2) locl hyperemi, inflmmtion nd oedem; (3) hyperemi, extensive oedem nd locl hemorrhge; (4) extensive hemorrhge; (5) locl necrosis nd pneumtosis intestinlis; (6) extensive necrosis nd pneumtosis intestinlis (15). NEC ws defined s score of minimum 3 in minimum of one region. Orgns (stomch, smll intestine, colon, liver, spleen, kidneys, lungs nd hert) were weighed, nd the tissues from three smll-intestinl regions nd from the liver were smpled nd snp-frozen for functionl nd immunologicl nlyses. Smples from the distl smll intestine were formldehydefixed nd prffin-emedded to investigte mucosl structures. Degree of lung expnsion ws determined s lung density (weight/volume), with volume recorded y wter displcement. Mucosl permeility, villus structure nd digestive function Intestinl permeility ws tested, s descried previously (15), s the post-mortem urine rtio of lctulose nd mnnitol fter orl dministrtion of the two sugrs ( 5 nd 3 g/kg, respectively) 4 h efore euthnsi. Dimensions of villi, crypts nd non-mucosl tissue (sumucos, lmin musculris nd seros) in the distl smll intestine were mesured (SoftWoRx Explorer version 1.1; Applied Precision, Issquh, WA, USA) on scnning pictures otined with n ArryWoRx e microrry scnner (Applied Precision). The ctivity of mucosl discchridses nd peptidses ws nlysed s descried previously (16). Systemic nd mucosl immune prmeters Plsm levels of the cute-phse protein, hptogloin, t irth, 6 h nd t euthnsi were determined y sndwich ELISA using n in-house mouse nti-porcine hptogloin monoclonl ctching ntiody nd commercil rit ntihumn hptogloin detection ntiody (DAKO A3; DAKO, Glostrup, Denmrk), s descried previously (19). Gene expression in the distl smll intestine nd liver ws investigted y n in-house immune-focused mrna Micro Arry POM4 chip with 373 oligonucleotides, representing more thn 25 different genes (pltform ccession no. GPL7576) following the procedures descried previously (2). In short, totl RNA ws extrcted (RNesy Midi Kit with TRIzol regent; Qigen, Bllerup, Denmrk) from the tissues from sline-injected formul-fed pigs (n 9), sline-injected colostrum-fed pigs (n 5) nd LPS-injected formul-fed pigs (n 8). RNA qulity nd quntity were determined on Nno Chip 6, Agilent 21 ionlyser (Agilent Technologies, Nærum, Denmrk) nd on Nnodrop ND-1 spectrophotometer (Sveen nd Werner AB, Limhmn, Sweden), respectively, efore cdna synthesis (QuntiTect Reverse Trnscription Kit; Qigen). Smples nd reference pool of ll smples were lelled (Oyster 55 nd 65, respectively) using 3DNA Arry 9 expression rry detection kits (Genisphere, Htfield, PA, USA). Hyridistion, wshing nd scnning were performed s descried previously (2). Imges were processed in GenePixPro 6. (Moleculr Devices Corportion, Sunnyvle, CA, USA) nd nlysed using Acuity 4 (Moleculr Devices Corportion), reveling pirwise differences in gene expression mong tretment groups nd etween sick nd helthy pigs. Tissue levels of TLR-4, IL-1 receptor-ssocited kinse-1 nd IkB were investigted y immunolotting nlysis s

4 Neontl gut response to fetl cteri 855 Tle 1. List of ntiodies used for Western lotting Trget protein (size) Type Dilution Mnufcturer TLR4 (89 kd) Mouse monoclonl 1:2 GenWy, Sn Diego, CA, USA IRAK1 (8 kd) Rit polyclonl 1:1 Snt Cruz Biotechnology, Inc., Snt Cruz, CA, USA IkB- (39 kd) Rit polyclonl 1:1 Cell Signling Technology, Beverly, MA, USA TLR, toll-like receptor; IRAK1, IL-1 receptor-ssocited kinse-1. descried previously (21) y seprtion on dentured SDS-PAGE gels (7 % for TLR-4 nd IL-1 receptor-ssocited kinse-1 (IRAK1), nd 15 % for IkB) nd overnight incution with primry ntiody (Tle 1) on nitrocellulose memrnes followed y 1 h incution with secondry ntiody (got nti-rit IgG horserdish peroxidse or got nti-mouse IgG horserdish peroxidse, 1:5; Snt Cruz Biotechnology, Inc.). Blots were visulised with the horserdish peroxidse sustrte (ECL-plus; Amershm Biosciences, Pisctwy, NJ, USA), scnned nd quntified y ImgeQunt 5. softwre (Moleculr Dynmics; Amershm Biosciences, Sunnyvle, CA, USA). Activity of NF-kB ws determined on nucler extrcts from the distl smll intestine using the TrnsAM p65 NF-kB Trnscriptions Fctor Assy Kit (Active Motif, Crlsd, CA, USA). Following the mnufcturer s protocol, nucler extrcts were otined, nd duplictes of 2 2 mg protein were dded into the ninety-six-well plte together with different dilutions of the mnufcturer s positive control. After incution with the NF-kB p65 ntiody, detection with secondry ntiody nd development, following the mnufcturer s protocol, sornce ws red on SpectrMx 34 microplte spectrophotometer (Moleculr Devices Corportion) t 45 nd 655 nm. Clcultions nd sttisticl nlyses All results re presented s mens with their stndrd errors. Group differences in NEC incidences were tested using Fisher s exct test (SAS/STAT version 9.1; SAS Institute, Cry, NC, USA). The PROC MIXED procedure of two-wy ANOVA of SAS, with tretment, diet nd sex s fixed vriles, nd pigs nd sows s rndom vriles, ws used to test the men differences mong tretment groups nd etween pigs with nd without NEC for ll remining prmeters. After the initil nlyses showing no effect of intrmusculr LPS injections, we used for the dditionl nlyses reduced model incorporting only diet (colostrum nd formul), sex nd LPS (with or without mniotic LPS dministrtion) s the fixed effects. mrna microrry dt were nlysed in Acuity 4. (Moleculr Devices Corportion) s descried erlier. Differences in the numer of differentilly regulted genes fter Acuity tretment comprisons were tested using Fisher s exct test (SAS). For ll nlyses, P¼ 5 ws used s the criticl level of significnce. Results Clinicl outcomes All five sows showed signs of lour 3 5 d fter the fetl surgery, leding to immedite cesren delivery. Survivl of the injected pigs ws high for four of the sows with only one or two stillorns, while eight of the fifteen pigs from one sow were stillorn. A totl of sixty vile pigs were included in the further study (Fig. 1). Gesttionl ge t delivery ws d mong the five litters, nd the men irth weight ws kg cross the litters. Cortisol levels t irth did not differ mong litters or tretment groups (73 2 (SEM 2 4) ng/ml). The men postntl lifetime ws 46 1 (SEM 1 2) h. The clinicl, structurl, functionl nd immunologicl prmeters shown in Tle 2 did not differ mong the groups of pigs tht received intrmusculr injections with sline or LPS for ny of the diet groups (colostrum nd formul). Hence, the intrmusculr LPS dose ppered to e too low to elicit ny significnt effects, nd for ech diet, we pooled pigs given intrmusculr LPS or sline in utero to form two lrger control groups ccording to diet (control-colostrum (control-c), n 11 nd control-formul (control-f), n 32; Fig. 1). The pigs given the higher LPS dose, LPS-formul (LPS-F, n 17), remined seprte group, s these pigs differed in severl clinicl nd functionl prmeters from the control-f nd control-c groups. At the time of tissue collection, none of the control-c pigs hd ny NEC lesions in the gstrointestinl orgns. NEC incidence in the LPS-F group ws 18 % (three out of seventeen) v. 38 % (twelve out of thirty-two) in the control-f group (P¼ 2), while the NEC lesion score (Fig. 2(A)) ws significntly reduced in the LPS-F v. control-f group (P, 5). The control-c pigs lost less weight fter irth (32 (SEM 19) g) thn the control-f (115 (SEM 13) g) nd LPS-F pigs (97 (SEM 12) g, oth P, 5) nd hd lower reltive liver weight (25 7 (SEM 6) v. 3 6 (SEM 1 1) g/kg (LPS-F) nd 3 8 (SEM 8) g/kg (control-f), oth P, 5). Otherwise, reltive orgn weights nd reltive length of the smll intestine did not differ mong the three groups. Lung density ws significntly higher in the control- F pigs thn in the control-c nd LPS-F pigs, which did not differ ( 83 (SEM 2) v. 77 (SEM 2) nd 78 (SEM 2) g/ml, respectively; Fig. 2(D)). Blood gs vlues t irth nd t 6 h were not different mong the three tretment groups, ut t euthnsi, pigs with NEC, compred with helthy pigs, showed mrked cidosis with low ph (7 1 (SEM 12) v (SEM 2)), low HCO 3 levels (9 (SEM 2 ) v (SEM 1 2) mmol/l), nd high lctte (9 2 (SEM 2 3) v. 1 6 (SEM 2) mmol/l) nd K levels (6 3 (SEM 1 1) v. 3 9 (SEM 1) mmol/l). At irth nd 6 h, only low hptogloin levels could e detected, ut the levels incresed therefter nd were significntly higher in formul-fed v. colostrum-fed pigs t 2 d (Fig. 2(E)).

5 856 M. S. Cilieorg et l. Tle 2. Selected vlues of clinicl, gut structurl nd functionl indices for pigs given intrmusculr injections with sline or lipopolyscchride (LPS) nd fed formul or colostrum (Men vlues with their stndrd errors) Tretment LPS-im-F Sline-im-F LPS-im-C Sline-im-C Men* SEM Men* SEM Men* SEM Men* SEM Weight loss (g) NEC score SI weight (g/kg) Colon weight (g/kg) Liver weight (g/kg) Lung density (g/ml) Sucrsek (U/g) Mltsek (U/g) Lctsek (U/g) ApNk (U/g) ApAk (U/g) DPP IVk (U/g) Villus{ (mm) ph Lctte** (mmol/l) HCO 3 ** (mmol/l) LPS-im-F, formul-fed pigs given intrmusculr LPS injections; sline-im-f, formul-fed pigs given intrmusculr sline injections; LPS-im-C, colostrum-fed pigs given intrmusculr LPS injections; Sline-im-C, colostrum-fed pigs given intrmusculr sline injections; NEC, necrotising enterocolitis; SI, smll intestine; ApN, minopeptidse N; ApA, minopeptidse A; DPP IV, dipeptidyl peptidse IV. * Men vlues were compred etween the two groups of formul-fed pigs nd etween the two groups of colostrum-fed pigs, nd none were found to e significnt (ANOVA). Loss of ody weight from irth to killing. Men lesion score cross five gstrointestinl regions. Orgn weight reltive to ody weight t killing. k Men vlues for the proximl, mid nd distl smll intestine. U¼mmol hydrolysed sustrte per min t 37 8C. { Villus height in the distl smll intestine. ** Vlues in the whole lood t euthnsi. Mucosl integrity, structure nd digestive function Intestinl permeility ws threefold higher in the control-f pigs, reltive to the control-c pigs (P¼ 8), with intermedite vlues for the LPS-F group (Fig. 2(C)). Villus height in the distl smll intestine showed the opposite trend with highest vlues in the control-c pigs, lowest vlues in the control-f pigs (P, 5 reltive to control-c) nd intermedite vlues in the LPS-F pigs (Fig. 2(B)). Likewise, the villus height:crypt depth rtio ws significntly higher in colostrum-fed pigs (control-c: 5 1 (SEM 4)) thn in formul-fed pigs (3 9 (SEM 4) in the control-f nd 4 3 (SEM 3) in the LPS-F groups, oth P, 1). Compred with helthy formul-fed pigs, formul-fed pigs with NEC hd shortened villi (2 (SEM 49) v. 531 (SEM 31) mm), lowered crypts (65 (SEM 1) v. 19 (SEM 5)) nd thinner lyer of non-mucosl tissue (88 (SEM 6) v. 15 (SEM 3) mm, ll P, 1). Except for sucrse ctivity, which ws highest in the LPS-F pigs ( 22 (SEM 3) v. 15 (SEM 2) U/g in the proximl region nd 36 (SEM 9) v. 23 (SEM 3) U/g in the middle region, oth P, 1) (where 1 U ¼ 1 mmol hydrolysed sustrte per min t 37 8C), ctivity of discchridses nd peptidses cross ll three regions of the smll intestine ws highest in colostrum-fed pigs (Fig. 3). LPS incresed the men lctse, minopeptidse A nd N ctivities of formul-fed pigs, ut the effects reched sttisticl significnce only for lctse ctivity in the proximl nd middle intestine (14 2 (SEM 1 9) for LPS-F pigs v (SEM 1 8) U/g for control-f pigs, P, 5). Mucosl immune prmeters Bsed on mrna microrry nlysis of the smll intestine, tretment group comprisons reveled pirwise differences in gene expression due to prentl tretments, postntl diet regimens nd disese sttes. Due to reltively high tissue complexity nd smll differences in expression levels, we judged tht the microrry nlysis ws too insensitive to detect quntittively the up- or down-regultion of individul genes. We therefore used the totl numer of differentilly expressed genes in the intestinl nd heptic tissue s the min outcome mesure for specific group comprisons. These nlyses showed tht type of diet (formul or colostrum) hd greter influence on the numer of differentilly expressed genes compred with the presence or sence of LPS tretment, s six intestinl genes were differentilly expressed etween the LPS-F nd control-f pigs, while thirty-three genes were differentilly regulted etween the control-c nd control-f groups (Fig. 4(A), P, 5). Minly genes involved in poptosis, inflmmtion nd cute-phse responses in the smll intestine were ffected y formul-feeding. LPS dministrtion ppered to compenste for the dverse effects of formul, since only seventeen intestinl genes were differentilly regulted etween the LPS-F nd control-c pigs

6 Neontl gut response to fetl cteri 857 (A) Score (B) Villus height (µm) , (C) Lctulose:mnnitol rtio (D) 85 Lung density (g/ml) (Fig. 4(A), P, 5). The fifty-five genes differentilly expressed etween pigs with nd without NEC showed tht the presence of intestinl lesions influenced immune gene regultion even more thn diets (Fig. 4(A)). Minly genes involved in poptosis, immune response nd metolism ccounted for this difference. The following genes, or groups of genes, were ffected when NEC-ffected intestines were compred with helthy formul-fed intestines: ngiopoietin 2, Bcl2-xL, (E) 25 Hptogloin (µg/ml) h 6h 48h Fig. 2. (A) Necrotising enterocolitis lesion score, (B) villus height in the distl smll intestine, (C) intestinl permeility s indicted y urinry lctulose:mnnitol rtio, (D) lung density nd (E) plsm hptogloin levels t irth, 6 h fter irth nd t the time of tissue collection. Vlues re mens, with their stndrd errors represented y verticl rs., Men vlues with unlike letters were significntly different etween groups (P, 5)., Control-formul;, intr-mniotic lipopolyscchride;, control-colostrum. 25 fss-ssociting deth domin, components in TLR pthwys, IL, trnsforming growth fctor 3 nd its receptor, protesome ctivtor, endothelil NO synthse, complement components, hptogloin, plsminogen ctivting fctor, trnsferrin, po 1, sugr trnsporters (GLUT nd sodium-glucose-linked trnsporter 1), minopeptidse A nd N, lctte dehydrogense-c nd ftty cid-inding protein. Tretment group differences in the numer of differentilly regulted genes in the liver were 2 Activity (U/g) , Sucrse, Mltse Lctse ApN ApA DPPIV Fig. 3. Brush-order enzyme ctivities cross three regions of the smll intestine. Vlues re mens, with their stndrd errors represented y verticl rs., Men vlues with unlike letters were significntly different etween groups (P, 5). ApN, minopeptidse N; ApA, minopeptidse A; DPP IV, dipeptidyl peptidse IV., Control-formul;, intr-mniotic lipopolyscchride;, control-colostrum. 1 U ¼ 1 mmol hydrolysed sustrte per min t 37 8C.

7 858 M. S. Cilieorg et l. fewer thn for the intestine, nd cute-phse proteins dominted (Fig. 4(B)). Only twelve liver genes were differentilly expressed etween helthy nd NEC formul-fed pigs, indicting miniml effects of NEC on heptic immune genes. None of the included cute-phse proteins ws differentilly regulted etween the LPS-F nd control-c pigs, neither in the intestine nor in the liver. Immunolots reveled significntly higher expression of TLR-4 in the control-c pigs compred with the two groups of formul-fed pigs, which did not differ (Fig. 5(A)). However, protein undnce of IkB, the inhiiting protein of the proinflmmtory trnscription fctor, NF-kB, ws higher in the control-c (14 7 (SEM 1 1) units) nd the LPS-F (16 4 (SEM 8) units) pigs. IL-1 receptor-ssocited kinse-1 (IRAK1) nd levels of ctivted NF-kB in nucler extrcts did not differ mong the three groups (Fig. 5(B)). Discussion Uterine infections during pregnncy hve een ssocited with incresed risk of pre-term irth, infection nd overll neontl moridity (1,22,23). Some studies hve reported Numer of differentilly regulted genes etween groups (A) (B) elevted NEC incidence following prentl infection, ut results hve een inconsistent (7 9). The difficulties in reching conclusions regrding the effects of cteril exposure efore irth my e explined y the different loction, type nd length of exposure in utero, nd the multitude of differences in clinicl vriles ex utero, such s feeding nd ntiiotic regimens. After irth, stimultion of the TLR-4 receptor y Grm-negtive cteri stimultes NEC development during the suckling period of rodents (24 28), while in neontl mice, Grm-negtive exposure my provide enhnced immunologicl tolernce (5). Thus, prentl LPS stimultion due to intruterine infections my ffect postntl NEC resistnce. Aginst this ckground, we hypothesised tht prentl exposure to LPS would enhnce postntl gut development nd NEC resistnce in pre-term pigs y providing etter tolernce to gut colonistion. Consistent with our hypothesis, we found tht LPS exposure for 3 5 d efore pre-term delivery modestly reduced the severity of NEC lesions, villus trophy, intestinl permeility nd dmge to rush-order enzyme ctivities following formul-feeding. The finding tht the effects were most pronounced in the proximl intestine indictes tht this region ws more exposed to swllowed LPS c d LPS-F v. control-c LPS-F v. control-f Control-F v. control-c NEC v. helthy Fig. 4. Different functionl groups nd totl numer of genes tht were differentilly expressed in the (A) smll intestine nd (B) liver tissue etween groups of pre-term pigs. The pirwise comprisons of gene expression etween prentl nd postntl tretments (control-formul (control-f), control-colostrum (control-c) nd intr-mniotic lipopolyscchride (LPS)-F), nd etween pigs with nd without necrotising enterocolitis (NEC), indicte the extent of tissue immune nd metolic responses to the vrious tretments. The numer of differentilly regulted genes etween pigs exposed to intr-mniotic LPS (LPS-F), nd ech of the other two groups, were significntly lower thn etween these two groups (control-c v. control-f, thirty-three differentilly expressed genes, P, 5 for oth comprisons) nd much lower thn the numer of differentilly regulted genes etween helthy pigs nd pigs with NEC (fifty-five differentilly regulted genes, P, 1). The numers of differentilly regulted genes in the liver etween the groups were lower nd not significntly different etween different comprisons.,,c,d Men vlues with unlike letters were significntly different etween comprisons (P, 5)., Apoptosis;, immune response;, inflmmtion;, cutephse response;, metolism;, signl trnsduction;, other.

8 Neontl gut response to fetl cteri 859 (A) Intensity volume (millions) (B) Intensity volume (millions) TLR-4 IRAK1 Iκ-B thn the more distl regions. Nevertheless, our nlyses of the numer of differentilly expressed intestinl immune-relted genes show tht formul-fed pigs exposed prentlly to luminl LPS were indeed ffected lso in the distl intestine. Hence, the numer of differentilly regulted genes for this group ws intermedite etween the numers for control formul- nd colostrum-fed pre-term pigs. The distl prt is usully the region mostly ffected y NEC lesions (16 18), possily due to the high density of immune cells. Our hypothesis tht n intr-mniotic stimultion with Grm-negtive LPS would induce immunologicl development of the immture gut epithelium ws prtly sed on the clinicl oservtions of decresed respirtory distress following prentl infections (7 9) nd on the oserved eneficil effects of LPS injections on lung mturtion nd surfctnt production in neworn lms (1,12,29). Intr-mniotic IL-1 dministrtion lso improved lung mturtion nd surfctnt production in fetl rits (3). In fetl lms, the LPS effects on the fetl lung were independent of rise in fetl cortisol levels (31), nd lso in the present study, circulting levels of cortisol did not differ mong groups. In lms, LPS induced oth systemic nd locl inflmmtion with incresed inflmmtory cells nd cytokines in the cord lood, mniotic fluid nd lveolr fluid (12). This occurred s direct result of luminl epithelil stimultion with cteril compounds since lung mturtion occurred only fter LPS injection into the mniotic cvity, trche or stomch, while systemic intr-peritonel LPS injections cused severe cidosis or deth (11). The responses in fetl lms were dose-dependent, nd doses elow 2 mg/kg resulted in inflmmtion without tissue or orgn * Fig. 5. Intestinl protein undnce for (A) toll-like receptor (TLR)-4, IL-1 receptor-ssocited kinse-1 (IRAK1) nd IkB in response to tretment or (B) the presence of necrotising enterocolitis (NEC) s determined y Western lot nlysis of intestinl extrcts. Vlues re mens, with their stndrd errors represented y verticl rs. Reltive mount of (C) ctive NF-kB mong tretment groups nd (D) etween pigs with nd without NEC from nucler extrcts, s determined y ELISA., Men vlues with unlike letters were significntly different etween tretment groups (P, 5). * Men vlues were significntly different etween pigs with nd without NEC (P, 5)., Control-formul;, LPS, intr-mniotic lipopolyscchride-formul;, control-colostrum;, helthy;, NEC. * (C) Totl protein/ml (D) Totl protein/ml NF-κB * mturtion (29), while fetl lms survived fter s much s 4 mg/kg into the mniotic cvity (31). In the present study, the LPS dose of 4 mg/kg ws proly not sufficient to elicit full inflmmtory, tolerogenic nd mturtionl response. This, comined with the vrile ingestion of the mniotic fluid y ech fetus, my explin tht the luminl LPS dministrtion hd limited nd somewht vrile effect in the present study. On the other hnd, the significnt effects on lung density vlues (reflecting incresed lveolr expnsion) nd intestinl structure nd function, prticulrly in the proximl intestine (e.g. sucrse, lctse nd villous integrity), support tht LPS hd physiologicl effects. Better control of the dose, length nd site of fetl LPS exposure will e importnt in future experiments to clrify how LPS dministrtion mimics the uterine, fetl nd neontl responses to Grm-negtive choriomnionitis. Systemic LPS injections re commonly used to induce NEC in experimentl NEC niml models (13,14,32). As our fetl pigs injected intrmusculrly with LPS did not differ from the sline control pigs, the chosen dose of 14 mg/kg LPS ppered insufficient to induce systemic inflmmtion nd ltered postntl development. Nevertheless, cidosis, incresed TNF- nd rin injury were the result of low LPS doses ( 5 15 mg, intrvenously) to fetl lms (33), while 1 mg/kg cused severe cidosis nd deth (11). In postntl pigs, 2 mg/kg induced mild cidosis (34), while higher doses to neworn pigs (2 mg/kg, intrvenously) resulted in decresed numers of leucocytes nd lesions similr to infnt NEC (13). We chose reltively low doses of LPS for oth dministrtion routes, considering tht injection of

9 86 M. S. Cilieorg et l. multiple fetuses lte in gesttion is ssocited with high risk of spontneous ortion. Spontneous signs of lour occurred for ll sows within 3 5 d of surgery, indicting tht the modest LPS stimultion, comined with fetl surgery, ws sufficient to dvnce prturition. Mternl nesthesi nd fetl surgery lone do not usully initite prturition, s shown in our mny erlier studies on fetl pigs t similr gesttionl ges (17,35,36). The present study confirms erlier oservtions tht cows colostrum stimultes gut development nd NEC resistnce (15,16), proly vi immunoregultory, ntiiotic or trophic compounds (37). The effects of colostrum-feeding, reltive to formul, were generlly greter thn those of prentl LPS tretment. Beneficil effects of colostrum were lso oserved in neworn rts where the mother s milk rpidly reduced the high intestinl NF-kB ctivity (38). On the other hnd, formul-feeding stimulted NEC nd mintined high TLR-4 nd NF-kB ctivity with low levels of the inhiitory protein IkB (38). In pre-term pigs, we hve not found consistent formul- nd NEC-induced up-regultion of intestinl TLR-4 expression, nd in the present study, TLR-4 protein ws elevted in the colostrum group. Likewise, mternl milk enhnced, rther thn reduced, the TLR-4-medited responses in dult nd fetl enterocytes nd leucocytes in vitro (39). Immunolotting reveled tht protein undnce of IkB, n inhiitory protein of the TLR-4-dependent NF-kB pthwy, ws elevted in the LPS-exposed nd colostrum-fed pigs. Hence, the exct role of TLR-4 protein nd mrna expression, nd TLR-4-medited inflmmtory pthwys in ssocition with cteril stimuli, pro-inflmmtory diets nd NEC remins controversil. The results of the microrry nlyses of gut nd liver tissues indicted tht diet exerted min influence on the inflmmtory processes, lthough LPS injections lso ppered to modify the numer of differentilly expressed genes. In generl, gene regultion ws less ffected y diet nd LPS exposure, thn y the presence of NEC, s ssessed y the numer of differentilly expressed genes. The semiquntittive nture of the microrry nlysis precluded ny detiled study of single genes. Intestinl genes relted to oth immune function nd metolic/digestive ctivities were ffected, nd the chnges in intestinl gene expression were ssocited with only few chnges in expression for the sme genes in the liver. Nevertheless, systemic cutephse response ws indicted y the incresed plsm hptogloin levels in formul-fed v. colostrum-fed pigs. The pig represents n elegnt niml model to investigte how the immture intestine is ffected y systemic or luminl cteril stimuli in utero. Extensive fetl mnipultions re possile in the lte gesttion, nd NEC develops spontneously fter pre-term irth nd formul-feeding. We hve shown tht LPS my enhnce intestinl mturtion, immunity nd NEC resistnce, lthough the effects t the present dose were modest compred with those of NEC-protective diet such s cows colostrum. More studies using different cteril stimuli nd vrying doses nd sites of cteril exposure re required to etter understnd how prentl cteril products my ffect the pre-term neonte, nd thus influence the clinicl interventions for infnts eing orn premturely following uterine nd fetl infections. Acknowledgements The present study ws supported y the Dnish Strtegic Reserch Council under the progrm of FØSU. We wish to thnk Bente Synnetsvedt, Axel Kornerup Hnsen, Mlene Birck nd Elin Skytte for their ssistnce with the niml procedures. We lso thnk Kristin Møller, Sophi Rsmussen, Jonn Amenuvor nd Thoms Boye Pihl for their technicl ssistnce with the RNA nlyses. The uthors hve no conflicts of interest. References 1. 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