Patent Application: US-43281903-A

Abstract:
a method of investigating chemical changes resulting from commensal microflora colonisation of mammalian intestine which comprises : a ) measuring gene expression in commensal bacterium - colonized and germ - free intestine of at least one gene ; and b ) identifying a gene from a ) that has at least a 2 - fold difference in expression level between commensal bacterium - colonized and germ - free intestine . the method selects genes for further evaluation , and gives rise to the development of prophylactic treatments .

Description:
in order to study at a molecular level , the changes in the intestine orchestrated by commensal bacteria , germ - free mice were colonised with commensal bacteria including bacteroides thetaiotaomicron , a prominent component of the normal mouse and human intestinal microflora . global intestinal transcriptional responses to colonization were delineated using high - density oligonucleotide arrays and the cellular origins of specific responses established by laser capture microdissection and real - time quantitative rt - pcr . the results illustrated hereinafter , reveal that commensal bacteria modulate expression of a large number of genes , some to significant levels . the genes involved participate in diverse and fundamental physiological functions of the gut , including nutrient absorption , mucosal barrier fortification , and xenobiotic metabolism . the species - selectivity of some of the colonization - associated changes in gene expression emphasizes how human physiology can be impacted by changes in the composition of indigenous microflora . furthermore , it would appear that some commensals play a role in postnatal developmental transitions . changes associated with the suckling - weaning transition were elicited in adult mice by b . thetaiotaomicron , suggesting that indigenous bacteria play an instructive role in postnatal gut development . coupling defined in vivo models with comprehensive genome - based analyses thus provides a powerful approach for identifying the critical contributions of resident microbes to host biology . [ 0092 ] bacteroides thetaiotaomicron is a genetically - manipulatable anaerobe and was chosen for initial study to define the impact of resident bacteria on intestinal biology because it is a prominent member of both the adult mouse and human gut microflora . moreover , b . thetaiotaomicron normally colonizes the distal small intestine ( ileum ) during the suckling - weaning transition , a time of rapid and pronounced functional maturation of the gut ( w . e . c . moore , l . v . holdeman , appl . microbiol . 27 , 961 ( 1974 ), t . ushijima , m . takahashi , k . tatewaki , y . ozaki , microbiol . immunol . 27 , 985 ( 1983 )). colonization elicited a concerted response involving enhanced expression of four components of the host &# 39 ; s lipid absorption machinery . mrnas encoding pancreatic lipase related protein - 2 ( plrp - 2 ) and colipase increased an average of 4 - and 9 - fold , respectively ( tables 1 and 2 ). plrp - 2 hydrolyzes tri - and diacylglycerols , phospholipids and galactolipids . colipase augments plrp - 2 activity ( m . e . lowe , m . h . kaplan , l . jackson - grusby , d . d &# 39 ; agostino , m . j . grusby , j . biol . chem . 273 , 31215 ( 1998 )). in addition , there was a 4 - 6 - fold increase in l - fabp mrna , which encodes an abundant cytosolic protein involved in fatty acid trafficking within enterocytes , and an induction of apolipoprotein aiv , a prominent component of triglyceride - rich lipoproteins ( chylomicrons , vldl ) secreted from the basolateral surfaces of enterocytes ( table 1 ). colonization led to an increase in ileal levels of na + / glucose cotransporter ( sglt - 1 ) mrna . there was also a concerted rise in expression of several components of the host &# 39 ; s lipid absorption / export machinery , including pancreatic lipase - related protein - 2 ( plrp - 2 ), colipase , liver fatty acid binding protein ( l - fabp ), and apolipoprotein a - iv ( see table 1 hereinafter ). the prominent decrease in expression of fasting - induced adipose factor , a novel pparα target known to be repressed with fat feeding ( s . kersten , et al ., j . biol . chem . 275 , 28488 ( 2000 ). ), provided further evidence for augmented lipid uptake in colonized mice . the changes in expression of these 6 genes in particular indicate that b . thetaiotaomicron elicits an increased host capacity for nutrient absorption / processing and may provide a partial explanation as to why germ - free rodents require a higher caloric intake to maintain their weight than those with a normal microflora ( b . s . wostmann , c . larkin , a . moriarty , e . bruckner - kardoss , lab . anim . sci . 33 , 46 ( 1983 ). additionally , the applicants have found that colonisation produces changes in expression of four genes involved in dietary metal absorption . a high affinity epithelial copper transporter ( crt1 ) mrna was increased , while metallothionein - i , metallothionein - ii , and ferritin heavy chain mrnas were decreased ( table 1 ). these changes suggest that colonization engenders increased capacity to absorb heavy metals ( e . g ., via crt1 ) and a concomitant decreased capacity to sequester them within cells ( mt - i / ii , ferritin ). this implies greater host demand for these compounds , either due to increased utilization by the host &# 39 ; s own metabolic pathways or to competition with the microbe . the changes in sglt - 1 , colipase , l - fabp , and mt1 ( plus 8 other mrnas discussed below ), were independently validated by qrt - pcr ( c . a . heid , j . stevens , k . j . livak , p . m . williams , genome res . 6 , 986 ( 1996 ) ( see table 2 below ). colipase plays a critical role in dietary lipid metabolism by stimulating the activity of both pancreatic triglyceride lipase and plrp - 2 . furthermore , proteolytic cleavage of procolipase yields a pentapeptide ( enterostatin ) that functions as a satiety signal for fat ingestion ( s . okada , d . a . york , g . a . bray , physiol . behav . 49 , 1185 ( 1991 )). the significantly elevated expression found following colonisation with b . thetoaiotaomicron illustrated hereinafter are indicative of a previously unappreciated mechanism by which the intestinal epithelium , together with a resident gut bacterium , contributes to dietary lipid metabolism . an intact mucosal barrier is critical for accomodating the vast population of resident intestinal microbes . its disruption can provoke an immune response that is deleterious to the host and to the stability of luminal microflora , leading to pathologic states such as inflammatory bowel disease ( p . g . falk , et al ., microbiol . mol . biol . rev . 62 , 1157 ( 1998 )). it has been found that colonization with b . thetaiotaomicron produces no detectable inflammatory response , as judged by histologic surveys ( l . bry , p . g . falk , t . midtvedt , j . i . gordon , science 273 , 1380 ( 1996 ). moreover , there is no discernible induction ( or repression ) of the many genes , represented on the microarrays , that are involved in responses . an influx of iga - producing b - cells does occur in the ileal mucosa 10 days after introduction of b . thetaiotaomicron ; similar commensal - induced iga responses have been shown to be t - cell independent and to enforce barrier integrity ( a . j . macpherson et al ., science 288 , 2222 ( 2000 ). genes involved in barrier function account for 10 % ( 7 / 71 ) of the changes in gene expression observed with b . thetaiotaomicron colonization . microarray and qrt - pcr analyses revealed that the influx of iga producing b - cells is accompanied by increased expression of the polymeric immunoglobulin receptor ( pigr ) that transports iga across the epithelium ( tables 1 , 2 ). there is also augmented expression of the crp - ductin gene , encoding both a component of the protective mucus layer overlying the epithelium ( muclin ; r . c . delisle , et al ., am . j . physiol . 275 , g219 ( 1998 )) and a putative receptor for trefoil peptides that participate in fortification / healing of the intestinal mucosa ( l . thim , e . mørtz , regul . pept . 90 , 61 ( 2000 ). additionally , there is increased expression of decay accelerating factor ( daf ), an apical epithelial surface protein that inhibits complement - mediated cytolysis ( m . e . medof , et al , j . exp . med . 165 , 848 ( 1987 ). coincident enhancement of pigr , muclin , and daf expression should not only help prevent bacteria from crossing the epithelial barrier , but should also prevent mucosal damage that may ensue from microbial activation of complement components present in intestinal secretions . it has been found that decay accelerating factor ( daf ) expression increased 5 - fold with colonization using b . thetaiotaomicron . daf is known to be present on the apical surface of intestinal epithelial cells and to inhibit complement - mediated cytolysis ( m . e . medof , e . i . walter , j . l . rutgers , d . m . knowles , v . nussenzeig , j . exp . med . 165 , 848 ( 1987 ),). the coincident enhancement of daf , pigr , and muclin expression should not only help prevent bacteria from crossing the epithelial barrier , but also prevent any mucosal damage that may ensue from microbial activation of complement components present in intestinal secretions . the most pronounced response to b . thetaiotaomicron was an increase in s mall pr oline - r ich protein - 2 ( sprr2a ) mrna ( table 1 ). qrt - pcr analysis established that there is a 205 ± 64 - fold elevation in this mrna with colonization ( table 2 ). sprr2a is a member of a family of proteins associated with terminal differentiation of squamous epithelial cells . sprrs contribute to the barrier functions of squamous epithelia , both as a component of the cornified cell envelope , and as cross - bridging proteins linked to desmosomal desmoplakin , a prominent desmosomal constituent ( p . m . steinert , l . n . marekov , mol . biol . cell 10 , 4247 ( 1999 ). colonization did not produce a notable change ( i . e . two fold or more ), in the expression of genes encoding other proteins linked to desmosomes ( desmoplakin , plakoglobin , plakophilin , plectin ), or tight junctions ( zo - 1 , occludin ). sprr2a expression in the intestine and its microbial regulation are novel findings . the critical contribution of sprr2a to the squamous epithelial barrier and the dramatic response of sprr2a expression to b . thetaiotaomicron together suggest that this protein plays an important role in intestinal barrier function . it is therefore a particularly suitable target for further investigation in accordance with the invention , in particular by evaluating the biochemical pathway in which sprr2a participates in the intestine . a prominent marker of the weaning transition is the decline in lactase - phlorizin hydrolase ( lph ), an enterocytic brush - border enzyme that hydrolyzes the principal milk sugar , lactose . lph mrna levels rise throughout the small intestine of conventionally raised animals during the suckling period , and then fall in the ileum during weaning ( s . d . krasinski et al ., am . j . physiol . 267 , g584 ( 1994 )). the effects of commensal bacteria on expression of these genes in particular may be of interest in determining whether the bacteria have signficant impact . using the method of the invention , it has been found that colonization results in increased expression of angiogenin - 4 which resembles angiogenin - 3 , a secreted protein with demonstrated angiogenic activity ( x . fu , et al ., mol . cell biol . 17 , 1503 ( 1997 ), x . fu , et al ., growth factors 17 , 125 ( 1999 )). the 11 - fold increase in expression of the angiogenesis factor recognizable by amplification using primers of seq id no 12 and seq id no 25 , which is angiogenin - 4 ( table 1 , 2 ) upon b . thetaiotaomicron colonization represents a novel mode of regulation for an angiogenesis factor and so may be the subject of further investigation in accordance with the invention . laser capture microdissection ( lcm ) experiments described below have delineated the cellular origins of this response . this suggests that the presence of bacteria influences intestinal vascularization . the gut is the site of first contact of innumerable ingested toxins and xenobiotics . the relative contributions of luminal bacteria and the epithelium to detoxification and metabolism of these compounds has been difficult to delineate in conventionally - raised mammals . it has been found that colonization of germ - free mice with b . thetaiotaomicron results in reduced expression of several genes involved in these processes ( table 1 below ). there is a decrease in the mrna encoding glutathione s - transferase , which detoxifies a variety of electrophiles , and a corresponding decrease in multi - drug resistance protein - 1 ( mdr - 1 ), which exports glutathione - conjugated compounds from the epithelium ( r . w . johnstone , a . a . ruefli , m . j . smyth , trends biochem . sci . 25 , 1 ( 2000 )). expression of cyp2d2 ( debrisoquine hydroxylase ) involved in oxidative drug metabolism in humans ( m . ingelman - sundberg , et al ., trends pharmacol . sci . 20 , 342 ( 1999 ), also declines with colonization . reduced expression of these genes indicates that b . thetaiotaomicron may inself contribute to detoxification of compounds that are deleterious to the host . a genetic polymorphism that produces a deficiency in this cytochrome p - 450 is common in humans and associated with altered oxidative drug metabolism ( m . ingelman - sundberg , m . oscarson , r . a . mclellan , trends pharmacol . sci . 20 , 342 ( 1999 )). the reduced expression of these three host genes suggests that commensal bacteria such as b . thetaiotaomicron contributes to the detoxification of compounds that could be deleterious to the host . this indicates that a component of the normal microflora can modulate host genes involved in drug metabolism , and underscore how variations in such metabolism between individuals may arise from differences in their resident gut flora . consequently , evaluation of the effect on commensal bacteria on expression of these genes using the method of the invention may be helpful pten is a member of a family of dual specificity protein phosphatases . pten haploinsufficiency in humans is associated with increased susceptibility to tumorigenesis ( d . j . marsh et al ., hum . mol . genet . 7 , 507 ( 1998 )). furthermore , pten +/−⇄ pten +/+ chimeric mice develop colonic polyps and adenocarcinoma ( a . dicristofano , b . pesce , c . cordon - cardo , p . p . pandolfi , nat . genet . 19 , 348 ( 1998 )). the human homolog of gp106 , tb2 / dp1 , is a component of a locus which when mutated produces multiple intestinal adenomas ( r . w . burt , adv . exp . med . biol . 470 , 99 ( 1999 )). the finding that a component of the microflora affects expression of genes such as the angiogenesis factor whose gene is amplifiable using primers such as seq id no 12 and 25 ( see table 3 hereinafter ) which is angiogenin - 4 , pten and gp106 highlights the importance of considering mechanisms by which intestinal bacteria may contribute to the initiation or progression of tumorigenesis within , or even outside , the gut . the motility of the intestine is regulated by its enteric nervous system ( ens ). the relative contributions of intrinsic and extrinsic factors to ens activity are poorly understood , despite the fact that irritable bowel syndrome , which involves dysregulated motor activity , is a major health problem . the impact of commensal bacteria such as b . thetaiotaomicron on gut physiology extends to genes expressed in the enteric nervous system ( ens ) and in the muscular layers . mrnas encoding the l - glutamate transporter and l - glutamate decarboxylase , which converts glutamate to gaba , are both increased , suggesting a colonization - associated effect on the glutamatergic neurons of the ens ( m . t . liu , j . d . rothstein , m . d . gershon , a . l . kirchgessner , j . neurosci . 17 , 4764 ( 1997 )). enhanced expression of vesicle - associated protein - 33 , a synaptobrevin - binding protein involved in neurotransmitter release ( p . a . skehel et al ., proc . natl . acad . sci . u . s . a . 97 , 1101 ( 2000 ) is also observed . there is a concomitant increase in two muscle - specific mrnas : enteric γ - actin and cysteine - rich protein 2 . previous electrophysiological studies of germ - free and conventionally - raised animals have suggested that the microflora plays a role in gut motility ( e . husebye , p . m . hellstrom , t . midtvedt , dig . dis . sci . 39 , 946 ( 1994 )). the method of the invention can provide molecular details about how resident gut microbes , such as b . thetaiotaomicron , may act to modulate motility . [ 0111 ] b . thetaiotaomicron normally colonizes mouse and human intestine during weaning ( w . e . c . moore , l . v . holdeman , appl . microbial . 27 , 961 ( 1974 ), t . ushijima , m . takahashi , k . tatewaki , y . ozaki , microbiol . immunol . 27 , 985 ( 1983 )). this period is characterized by a dramatic shift in the composition of the microflora and by a series of critical developmental changes in the intestinal epithelium . it is unclear how many of these changes are regulated by intrinsic cellular mechanisms , and how many are controlled by extrinsic signals emanating from the mesenchyme , or from luminal ( dietary , microbial ) factors . expression profiling revealed surprisingly that colonization of adult germ - free mice with b . thetaiotaomicron elicits other responses that mimic changes which normally occur in the maturing intestine of conventionally - reared animals . expression of lactase , which hydrolyzes the principal milk sugar ( lactose ), normally declines during the weaning period ( s . d . krasinski et al ., am . j . physiol . 267 , g584 ( 1994 ). colonization of adult germ - free mice with b . thetaiotaomicron produces a decrease in ileal lactase mrna ( table 1 , 2 below ). these findings indicate how members of the emerging postnatal normal flora may contribute to intestinal maturation . adenosine deaminase ( ada ) and polyamines ( spermine , spermidine ) play important roles in postnatal intestinal maturation ( g . d . luk , l . j . marton , s . b . baylin , science 210 , 195 ( 1980 ), j . m . chinsky , et al ., differentiation 42 , 172 ( 1990 )). it has been found that b . thetaiotaomicron colonization produces an increase in mrnas encoding ada and ornithine decarboxylase ( odc ) antizyme but not a 5 - fold increase . the antizyme , whose expression is affected by polyamine levels , is a critical regulator of odc turnover ( j . nilsson , s . koskiniemi , k . persson , b . grahn , i . holm , eur . j . biochem . 250 , 223 ( 1997 )); an increase in antizyme mrna levels therefore suggests that colonization influences ileal polyamine synthesis . these data demonstrate that genes controlling synthesis of two classes of regulators of gut maturation , adenosine and polyamines , are themselves modulated by a component of the microflora , leading to the idea that bacteria serve as upstream effectors of a cascade that affects gut maturation . other colonization experiments , described below , indicate that other gut commensals have the capacity to engineer such changes . changes in gut maturation associated with the suckling - weaning transition are also thought to be regulated by increases in glucocorticoids ( s . j . henning , d . c . rubin , r . j . shulman , in physiology of the gastrointestinal tract , l . r . johnson , ed . ( raven press , new york , 1994 ), pp . 584 - 586 ). b . thetaiotaomicron colonization as described hereinafter was accompanied by reduced expression of two genes whose transcription is known to be suppressed by glucocorticoids : 15 - hydroxyprostaglandin dehydrogenase ( m . d . mitchell , v . goodwin , s . mesnage , j . a . keelan , prostaglandins leukot . essent . fatty acids 62 , 1 ( 2000 )) and glucocorticoid - attenuated response gene - 16 ( j . b . smith , h . r . herschman , j . biol . chem 270 , 16756 ( 1995 )). furthermore , there was reduced expression of another gene whose product interacts with nuclear hormone receptor family members , the immunophilin fkbp51 ( s . c . nair et al ., mol . cell . biol . 17 , 594 ( 1997 )). however , the reduction was not greater than 5 - fold in any individual case . thus , other commensal bacteria may be investigated using the method of the invention to see if these could produce a more significant effect . as mentioned above , the applicants have found that a particular member of the angiogenin family , whose gene is amplifiable using primers of seq id no 12 and 25 above and is expressed in mouse intestine , is novel . thus this protein and the gene encoding it forms a further aspect of the invention . a further aspect of the invention provides a protein of seq id no 29 as shown in fig4 hereinafter , or an allelic variant thereof or a protein which has at least 85 % amino acid sequence identity with seq id no 29 . in particular , the invention provides a protein of seq id no 29 . in yet a further aspect , the invention provides a nucleic acid which encodes a protein as described above . these proteins are useful as a target for the screening process of the invention . age - matched groups of 7 - 15 week - old germ - free nmri / ki mice were maintained in plastic gnotobiotic isolators on a 12 hour light cycle , and given free access to an autoclaved chow diet ( b & amp ; k universal ). males were inoculated with wild - type b . thetaiotaomicron ( strain vpi - 5482 ) ( l . hooper et al ( 1999 ) supra ). mice were sacrificed 10 days later , 2 hours after lights were turned on . the distal 1 cm of the small intestine was used to define the number of colony forming units per ml of extruded luminal contents . ileal rna was isolated from mice with & gt ; 10 7 colony forming units ( cfu ) of bacteria per ml of luminal contents . [ earlier studies had shown that 10 days was sufficient to produce robust colonization of the ileum and that = 10 7 cfu / ml were necessary for full induction of fucosylated glycan production in the ileal epithelium ( l hooper et al , ( 1999 ) supra . l . bry , p . g . falk , t . midtvedt , j . i . gordon , science 273 , 1380 ( 1996 ))]. total ileal rna samples , prepared from the 3 cm of intestine adjacent the distal 1 cm of the small intestine of 4 mice from 3 independent colonizations , and from age - and gender - matched germ - free mice ( n = 8 ), using a rna ( qiagen rneasy kit ), were each pooled , in equal amounts , for generation of biotinylated crna targets . two targets were prepared , independently , from 30 μg of each total cellular rna pool , using the method outlined by c . k . lee , et al ., science 285 , 1390 ( 1999 ). sybr green - based real - time quantitative rt - pcr studies ( n . steuerwaldet al ., mol . hum . reprod . 5 , 1034 ( 1999 )) were performed using the gene - specific primers listed in table 3 below and dnase - treated rnas . control experiments established that the signal for each amplicon was derived from cdna and not from primer dimers or genomic dna . signals were normalized to an internal reference mrna ( glyceraldehyde 3 - phosphate dehydrogenase ). the normalized data were used to quantitate the levels of a given mrna in germ - free and colonized ileums ( δδc t analysis ; bulletin # 2 , abi prism 7700 sequence detection system ). each crna was hybridized to affymetrix mu11k and mu19k chip sets representing ˜ 25 , 000 unique mouse genes from unigene build 4 and the tigr cluster databases , according to affymetrix protocols . data collected from each chip were scaled so that the overall fluorescence intensity across each chip was equivalent ( target intenstity = 150 ). pairwise comparisons of ‘ germ - free ’ versus ‘ colonized ’ expression levels were performed . a 2 - fold or more difference was recorded if three criteria were met : the genechip software returned a difference call of “ increased ” or “ decreased ”, the mrna was called ‘ present ’ by genechip software in either germ - free or colonized crna , and the difference was observed in duplicate microarray hybridizations . mrnas represented by 118 probe sets changed by at least 2 - fold with colonization , as defined by duplicate microarray hybridizations . it was found that transcripts represented by 95 probe - sets were increased , while those represented by 23 probe - sets were decreased . the genes represented by 84 of these probe sets ( 71 unique genes ) were assigned to functional groups and these are set out in table 1 hereinafter . in this table , results are presented as the fold - difference in mrna levels between colonized and germ - free ileum and represent average values from duplicate microarray hybridizations . the average fold - changes for genes represented by 2 or more independent probe sets are listed separately . importantly , a large of number of the genes identified using these criteria are involved in modulating fundamental intestinal functions : 20 of the 71 genes ( 28 %) were grouped under nutrient uptake and metabolism . there was also a concerted rise in expression of several components of the host &# 39 ; s lipid absorption / export machinery , including pancreatic lipase - related protein - 2 ( plrp - 2 ), colipase , liver fatty acid binding protein ( l - fabp ), and apolipoprotein a - iv ( table 1 ). the prominent . decrease in expression of fasting - induced adipose factor , a novel pparg target known to be repressed with fat feeding ( s . kersten , et al ., j . biol . chem . 275 , 28488 ( 2000 ), provided further evidence for augmented lipid uptake in colonized mice . the changes in expression of these 6 genes indicate that b . thetaiotaomicron elicits an increased host capacity for nutrient absorption / processing and helps explain why germ - free rodents require a higher caloric intake to maintain their weight than those with a microflora . additionally , there were changes in expression of four genes involved in dietary metal absorption . a high affinity epithelial copper transporter ( crt1 ) mrna was increased , while metallothionein - i , metallothionein - ii , and ferritin heavy chain mrnas were decreased ( table 1 ). these changes suggest that colonization engenders increased capacity to absorb heavy metals ( e . g ., via crt1 ) and a concomitant decreased capacity to sequester them within cells ( mt - i / ii , ferritin ). this implies greater host demand for these compounds , either due to increased utilization by the host &# 39 ; s own metabolic pathways or to competition with the microbe . the changes in sglt - 1 , colipase , l - fabp , and mt1 ( plus 8 other mrnas discussed below ), were independently validated by qrt - pcr ( c . a . heid , j . stevens , k . j . livak , p . m . williams , genome res . 6 , 986 ( 1996 ). ( table 2 ). of these , genes which were found to have a difference in expression levels of 5 - fold or more as a result of b . thetaiotaomicron colonisiation were colipase , liver fatty acid binding protein , fasting - induced adipose factor , metallothionein i and metallothionein ii , malate oxidoreductase , sprr2a , angiogenin - 3 , angiogenin - related protein , angiogenin family , gelsolin , gp106 ( tb2 / dp1 ) and rac 2 . of these , colipase , fasting - induced adipose factor , angiogenin 3 and sprr2a genes showed a difference in expression levels of 9 - fold or more . a notable feature of the host response to b . thetaiotaomicron was the absence of detectable or changed expression of the many genes involved in immuno - inflammatory processes that are represented on the microarrays . these include genes involved in the nf - κb - regulated processes that are critical regulators of host responses to invasive pathogens ( d . elewaut et al ., j . immunol . 163 , 1457 ( 1999 )). the absence of these responses can be contrasted to results obtained in a recent cdna microarray analysis of the response of a human intestinal epithelial cell line to salmonella , an invasive gut pathogen ( l . eckmann , j . r . smith , m . p . housley , m . b . dwinell , m . f . kagnoff , j . biol . chem . 275 , 14084 ( 2000 )). the lack of evidence for an evoked in vivo immuno - inflammatory response is consistent with the host &# 39 ; s need to accommodate resident gut microbes , such as b . thetaiotaomicron , for its entire lifespan . colonization increases expression of two genes implicated in development of gut neoplasia , pten and gp106 ( table 1 ). in a further analysis two techniques were combined . first , laser - capture microdissection ( lcm ) was used to recover three cell populations from frozen sections of ileum harvested immediately after sacrifice of germ - free and colonized mice . the three populations are ( i ) epithelium present in crypts ( the proliferative compartment of the intestine containing undifferentiated cells as well as differentiated members of the paneth cell lineage ); ( ii ) epithelium overlying villi ( containing post - mitotic , differentiated members of the intestine &# 39 ; s other three lineages ); and ( iii ) mesenchyme underlying crypt - villus units ( fig1 ). lcm was performed on groups of mice independent of those used to generate rna for the microarray analysis . 7 μm - thick sections were cut from frozen ileums and lcm conducted using the pixcell ii system from arcturus ( 7 . 5 μm diameter laser spot ). rna was prepared from dissected cell populations using the rna micro - isolation kit ( strategene ) and standard histochemical protocols . laser capture microdissection ( lcm ) was carried out using conventional methods as described by m r emmert - buck et al ., science ., 274 , 998 - 1001 , 1996 and r . f . bonner et al ., science 278 : 1203 - 4 , 1997 . second , real - time rt - pcr was used to quantitate levels of specific mrnas in the laser captured cell populations . the lcm / qrt - pcr analysis was performed using germ - free and colonized mice from 3 experiments that were independent of those used for microarray profiling . each sample was analyzed in triplicate in 4 - independent experiments . mean values for the independent determinations ± 1 s . d . are shown in table 2 colipase is produced by the exocrine ( acinar ) cells of the pancreas . expression in the intestine had not been reported previously . therefore , lcm and real - time rt - pcr analysis were employed to delineate the cellular origins of its response to b . thetaiotaomicron . the results show that sprr2a mrna is confined to the epithelium where its concentration is 7 - fold higher on the villus compared to the crypt ( fig1 b ). b . thetaiotaomicron elicits a 280 - fold increase in the villus epithelium . this value is in good agreement with the increase documented in total ileal rna ( table 2 ). the cellular origin of the sprr2a response supports the hypothesis that it participates in fortifying the intestinal epithelial barrier in response to bacterial colonization . colipase is produced by the exocrine acinar cells of the pancreas . lcm / qrt - pcr revealed that colipase mrna is also present in the ileal crypt epithelium , where it increases 10 - fold upon b . thetaiotaomicron colonization ( fig1 b ). this accounts for the increase detected by microarray and qrt - pcr analyses of total ileal rna ( tables 1 , 2 ). colipase plays a critical role in dietary lipid metabolism by stimulating the activity of both pancreatic triglyceride lipase and plrp - 2 ( m . e . lowe , et al ., j . biol . chem . 273 , 31215 ( 1998 ). furthermore , proteolytic cleavage of procolipase yields a pentapeptide ( enterostatin ) that functions as a satiety signal for fat ingestion ( s . okada , et al ., physiol . behav . 49 , 1185 ( 1991 ). analyses of colipase gene regulation reveal a previously unappreciated contribution of the intestinal epithelium ( together with a resident gut commensal ) to dietary lipid metabolism . angiogenin - 3 was originally identified in nih 3t3 fibroblasts ( x . fu et al ., mol . cell biol . 17 , 1503 ( 1997 ), but little is known about its cellular origins or regulation . lcm and qrt - pcr revealed that the crypt epithelium is the predominant location of a gene amplifiable using primers such as seq id no 12 and 25 ( see table 3 hereinbefore ) which are specific for angiogenin - 3 mrna , but also for the new protein which is angiogenin - 4 mrna and that colonization results in a 7 - fold increase in its levels within this compartment . this increase accounts for the change in expression defined by microarray and qrt - pcr analyses of total ileal rna ( tables 1 , 2 ). the epithelial location of a secreted / rnase / angiogenesis factor puts it in a strategic position to function as an effector of a number of host responses to microbial colonization ( e . g ., enhanced absorption / distribution of nutrients / augmented barrier function . the lcm / qrt - pcr studies of sprr2a , colipase and angiogenin - 4 establish the feasibility of assigning an in vivo host response to a particular cell population in a complex tissue , and of describing the cellular response in quantitative terms . in recovering a responding cell population and expressing its reaction to a microorganism in quantitative terms , the applicants results demonstrate how it is possible to move beyond in vitro models and use in vivo systems to study the impact of a microbe on host cell gene expression . colonization of germ - free mice with b . thetaiotaomicron produces a decrease in ileal lph mrna levels ( table 1 , 2 ) ( although not by as much as five times ). analysis of rna isolated from laser - captured epithelial and mesenchymal cell populations established that the colonization - induced reduction in lph mrna levels occurs primarily within the villus epithelium ( fig2 ). comparison of transcript levels between germ - free and b . thetaiotaomicron - associated mice revealed a colonization - associated increase in expression of angiogenin - 4 . the 11 - fold induction of its mrna seen in example 1 was independently validated by real - time rt - pcr of total ileal rnas ( table 2 ). angiogenin - 3 was originally identified in nih 3t3 fibroblasts ( x . fu , m . p . kamps , mol . cell biol . 17 , 1503 ( 1997 )). however , lcm and real - time rt - pcr analysis revealed that in colonized ileum , the levels of mrna which are amplifiable using primers designed for angiogenin - 3 are highest in crypt epithelium ( values in the ileal villus epithelium and mesenchyme are 14 - and 15 - fold lower , respectively ; fig2 ). as outlined in example 4 below however , this mrna is in fact , angiogenin - 4 mrna . the 7 - fold increase in these angiogenin - 4 mrna levels observed in the crypt epithelium after colonization account for the change defined by microarray and real - time rt - pcr analyses of total ileal rna . bacterial modulation of epithelially - expressed angiogenin - 4 represents a novel mode of regulation for an angiogenesis factor . lcm / qrt - pcr established that colonization reduces lactase mrna levels within the villus epithelium ( fig1 b ). the concept that microbes may help legislate changes in expression of developmentally - regulated genes , such as lactase , raises the question of whether some or many components of the microflora can elicit these changes . in order to examine this , age - matched groups ( n = 4 - 8 mice / group ) of 7 - 15 week - old germ - free nmri / ki mice were maintained in plastic gnotobiotic isolators on a 12 hour light cycle , and given free access to an autoclaved chow diet ( b & amp ; k universal ). males were inoculated with one of the following groups ( ii ) b . thetaiotaomicron strain vpi - 5482 ( l . v . hooper , et al ., proc . natl . acad . sci . u . s . a . 96 , 9833 ( 1999 )), ( iii ) e . coli k12 which was originally recovered from a normal human fecal flora , ( iv ) bifidobacterium infantis ( atcc15697 ), a prominent component of the pre - weaning human and mouse ileal flora and a commonly used probiotic . ( v ) a ‘ complete ’ ileal / cecal microflora harvested from conventionally - raised mice ( l . bry , et al ., science 273 , 1380 ( 1996 ) mice were sacrificed 10 days later , 2 hours after lights were turned on . the distal 1 cm of the small intestine was used to define cfu / ml ileal contents . the 3 cm of intestine just proximal to this segment was used to isolate total ileal rna ( qiagen rneasy kit ). qrt - pcr was used to compare ileal lactase mrna levels in each group ( all animals had = 10 7 cfu / ml ileal contents ). the results are shown in fig3 . colonization with any of the three gram - negative anerobes elicited an equivalent decline in lactase expression relative to germ - free controls ( fig3 ). this decline was also observed after inoculation of a complete ileal / cecal flora . qrt - pcr of the same rnas revealed that ileal expression of colipase and angiogenin - 4 was induced after colonization of all three organisms , and by the ileal / cecal flora ( fig3 ). the levels of colipase and angiogenin - 4 mrnas achieved in the ileums of these ex - germ - free mice were comparable to those of age - matched mice that have been conventionally - raised since birth ( fig3 ). in contrast to these findings , the response of sprr2a to colonization was dependent upon the colonizing species . while b . thetaiotaomicron produced a pronounced rise in sprr2a mrna that recapitulates the response to a 10 day colonization with the ileal / cecal flora , colonization with b . infantis and e . coli produce only negligible increases in mrna levels ( fig3 ). mdr1a and glutathione - s - transferase , which act in concert to metabolize xenobiotics and electrophiles , also exhibited species - specific ( and concerted ) responses . unlike b . thetaiotaomicron , which suppresses expression , e . coli and b . infantis both elicit increases in these mrnas . in contrast , the multi - component ileal / cecal flora did not produce a significant ( i . e .,= 2 - fold ) change in levels of either mrna when compared to germ - free controls . the mdr1a / gst responses provide direct evidence that components of the normal microflora can modulate host genes involved in drug metabolism , and suggest that variations in drug metabolism between individuals may arise , in part , from differences in their resident gut flora . following the observation that a 10 d colonization was associated with a 11 - fold increase in ileal expression of a mrna detected by an affymetrix - designed probe - set designed from the published sequence of angiogenin - 3 , we designed primers specific for the 3 ′ and 5 ′ ends of the mouse angiogenin - 3 . there were : these primers were used together with rt - pcr to amplify a 438 bp sequence from rna prepared from the ileums of ex - germ - free nmri mice . these mice had been colonized for 10 d with a complete ileal / cecal flora harvested from conventionally - raised animals belonging to the same inbred strain . we subcloned the pcr product into bamhi / xbai digested pgex - kg and sequenced it using vector - specific primers . surprisingly , the nucleotide sequence of the orf was only 90 % identical to that of mouse angiogenin - 3 . since the primer sequences used in the pcr reaction ( specific for angiogenin - 3 ) were incorporated into the product , we used 5 ′- and 3 ′- race to ( a ) obtain accurate sequence at the 5 ′ and 3 ′ ends of the orf of this new angiogenin , and ( b ) characterize the 5 ′- and 3 ′ untranslated regions of its mrna . the results revealed only 88 . 3 % nucleotide sequence identity with angiogenin - 3 mrna . the nucleotide sequence which encodes the angiogenin - 4 protein , aligned with the angiogenin - 3 sequence is shown hereinafter in fig4 as seq id no 29 and 30 respectively . angiogenin - 4 has 74 to 81 % amino acid sequence identity to the other 3 members of the mouse angiogenin family ( fig5 ). it was found that the 5 ′ and 3 ′- untranslated regions of angiogenin - 4 are closely related to the corresponding regions of angiogenin - 3 mrna ( fig4 ). subsequently a comparative analysis of the tissue distribution of the various mouse angiogenin mrnas , was conducted . cdna was synthesized from rnas isolated from tissues harvested from conventionally raised adult ( 12 - 14 week old ) male and female nmri mice ( 25 tissues / mouse ). to quantitate relative levels of expression of each gene , we designed primer sets specific for each of the four mouse angiogenin family members ( fig6 ; table 4 ) and used them for sybr - green - based real - time quantitative rt - pcr ( qrt - pcr ) analyses . remarkably , angiogenin - 4 mrna was restricted the intestine where it is expressed from the duodenum to the rectum ( fig7 ). in contrast , angiogenin - 1 expression is highest in liver , lung , and pancreas ( fig8 ), while angiogenin - 3 is expressed primarily in liver , lung , pancreas , and prostate ( fig9 ). angiogenin - related protein mrna was undetectable in all tissues surveyed even after 40 cycles of pcr ( fig1 ). thus , the highly restricted , intestine - specific pattern of angiogenin - 4 expression makes it unique among mouse angiogenin family members . these findings indicated that there was microbial - regulation of angiogenin - 4 rather than angiogenin - 3 expression in the intestine . to test this hypothesis directly , angiogenin - 4 - specific primers and qrt - pcr were used to compare angiogenin - 4 mrna levels along the length of the small intestine of germ - free nmri mice and germ - free mice colonized for 10 d with an ileal / cecal flora harvested from conventionally raised nmri animals . pair - wise comparisons revealed that expression of angiogenin - 4 is highest in the jejunum of colonized mice , and that conventionalization induces up to a 17 - fold increase in angiogenin - 4 expression in this region ( fig1 ). mono - association of germ - free nmri mice with b . thetaiotaomicron for 10 d resulted in a comparable induction of angiogenin - 4 expression ( data not shown ). the developmental patterns of angiogenin - 4 expression in postnatal day 5 ( p5 )— p30 germ - free and conventionally raised nmri mice ( n = 3 mice per time point per group ) was then assessed ( fig9 ). relative levels of the angiogenin - 4 transcript remained relatively low until p20 in both groups of mice . expression rose slightly ( 2 - 3 fold ) in germ - free animals after this time point . in contrast , angiogenin - 4 expression increased more than 20 - fold between p15 and p30 in conventionally - raised animals . these results indicate that angiogenin - 4 is induced during the suckling / weaning transition — coincident with a major shift in the gut microbiota . the lack of angiogenin - 4 induction in postnatal germ - free mice is also consistent with the conclusion that components of the microbiota play an important role in regulating angiogenin - 4 expression . the previous laser capture microdissection ( lcm )/ qrt - pcr study of the cellular origins of angiogenin protein expression ( example 2 ) used primers that recognize both angiogenin - 3 and angiogenin - 4 , and rnas that had been isolated from captured crypt epithelium , villus epithelium , or mesenchymal populations from the villus core . the qrt - pcr analysis indicated that the microbially - regulated ‘ angiogenin ’ was produced in epithelial cells located at the base of crypts of lieberkuhn ( hooper et al ., 2001 ). to test the hypothesis that angiogenin - 4 expression occurs in paneth cells , we used lcm to isolate cells located at the base of jejunal crypts from ( a ) germ - free adult ( 12 week old ) transgenic mice with an attenuated diphtheria toxin - a fragment ( tox176 )- mediated paneth cell lineage ablation ( cr2 - tox176 mice ) ( garabedian et al ., 1997 ), and ( b ) their age and gender - matched germ - free normal littermates . qrt - pcr using angiogenin - 4 - specific primers revealed that angiogenin - 4 mrna levels are 10 - fold higher in rna purified from crypt base epithelial cells of normal mice compared to cr2 - tox176 littermates ( fig1 ). a follow - up study was conducted using conventionally raised nmri mice . three cellular pools were harvested by lcm : paneth cells alone , epithelial cells from the upper crypt and villus ( a paneth cell - minus fraction ), and mesenchyme retrieved from the villus core and the peri - cryptal region . the distribution of angiogenin - 4 mrna closely paralleled the distribution of phospholipase a2 — the product of the mom - 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