Research Paper Gut Microbes 5:2, 152–164; March/April 2014; © 2014 Landes Bioscience

Anand Kumar1, Anastasia N Vlasova1, Zhe Liu1, Kuldeep S Chattha1, Sukumar Kandasamy1, Malak Esseili1, Xiaoli Zhang2, Gireesh Rajashekara1,*, and Linda J Saif1,* Food Animal Health Research Program; Department of Veterinary Preventive Medicine; Ohio Agricultural Research and Development Center; The Ohio State University; Wooster, OH USA; 2Center for Biostatistics; The Ohio State University; Columbus, OH USA

1

Keywords: lactobacilli, probiotics, transcriptome, gnotobiotic pigs, gut Abbreviations: LGG, Lactobacillus rhamnosus; LA, Lactobacillus acidophilus; Gn pigs, gnotobiotic pigs; PBCD, post bacterial colonization day; CFU, colony forming unit; IPA, Ingenuity pathway analysis; IPKB, Ingenuity pathways knowledge base; MLN, mesenteric lymph node; CP, canonical pathway

Probiotics facilitate mucosal repair and maintain gut homeostasis. They are often used in adjunct with rehydration or antibiotic therapy in enteric infections. Lactobacillus spp have been tested in infants for the prevention or treatment of various enteric conditions. However, to aid in rational strain selection for specific treatments, comprehensive studies are required to delineate and compare the specific molecules and pathways involved in a less complex but biologically relevant model (gnotobiotic pigs). Here we elucidated Lactobacillus rhamnosus (LGG) and L. acidophilus (LA) specific effects on gut transcriptome responses in a neonatal gnotobiotic (Gn) pig model to simulate responses in newly colonized infants. Whole genome microarray, followed by biological pathway reconstruction, was used to investigate the host-microbe interactions in duodenum and ileum at early (day 1) and later stages (day 7) of colonization. Both LA and LGG modulated common responses related to host metabolism, gut integrity, and immunity, as well as responses unique to each strain in Gn pigs. Our data indicated that probiotic establishment and beneficial effects in the host are guided by: (1) down-regulation or upregulation of immune function-related genes in the early and later stages of colonization, respectively, and (2) alternations in metabolism of small molecules (vitamins and/or minerals) and macromolecules (carbohydrates, proteins, and lipids). Pathways related to immune modulation and carbohydrate metabolism were more affected by LGG, whereas energy and lipid metabolism-related transcriptome responses were prominently modulated by LA. These findings imply that identification of probiotic strain-specific gut responses could facilitate the rational design of probiotic-based interventions to moderate specific enteric conditions.

Introduction Enteric pathogens cause diarrheal diseases in both adults and infants, with the latter being more susceptible due to an immature gut with increased permeability to antigens, as well as an immature immune system.1,2 Given this scenario, particularly in developing countries where availability of enteric vaccines or lower efficacy of vaccines (e.g., rotavirus vaccine) are problems, other means of treatment in addition to oral rehydration are often required. Probiotics that facilitate mucosal repair and maintain gut homeostasis are invaluable treatment modalities for neonatal enteric infections including rotavirus diarrhea.3 Recent research has highlighted the

effective role of probiotics in modulation of the gut microbiota, thereby alleviating intestinal disorders like inflammatory bowel disease, travelers’ diarrhea, colitis, Crohn disease, and antibiotic associated diarrhea.4,5 However, pathways and mechanisms by which probiotics mediate their beneficial effects, especially in neonates, are largely undefined. Among several probiotic bacteria, Gram-positive bacteria dominate, especially ones belonging to the Lactobacillus and Bifidobacterium genera. Lactobacillus species are probably the most widely consumed and marketed in the world because of the long history of safety in the food and dairy industry.6 However, overall they comprise only a small portion of the natural gut microflora.7 Many Lactobacillus spp. have been

*Correspondence to: Gireesh Rajashekara; Email: [email protected]; Linda J Saif; Email: [email protected] Submitted: 08/29/2013; Revised: 12/18/2013; Accepted: 01/15/2014; Published Online: 01/22/2014 http://dx.doi.org/10.4161/gmic.27877 152

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In vivo gut transcriptome responses to Lactobacillus rhamnosus GG and Lactobacillus acidophilus in neonatal gnotobiotic piglets

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genes were pronounced in LA-colonized Gn pigs. Collectively, our study provides new insights to the distinct and common cellular pathways modulated in Gn pigs by LA and LGG.

Results Colonization begins at early stage (PBCD1) and increases at later stage (PBCD7) On PBCD1, LA colonized duodenum (1.0 × 103 CFU/g), jejunum (9.4 × 102 CFU/g), and ileum (3.3 × 104 CFU/g) with higher CFU counts in the large intestine, reaching a maximum in the cecum (1.8 × 105 CFU/g). Surprisingly, LA was also detected in the spleen, liver, and MLNs in two of the three pigs. On the other hand, for LGG on PBCD1, the CFU counts were one log higher than LA in duodenum (2.6 × 104 CFU/g) and ileum (2.8 × 105 CFU/g), and the colonization in jejunum was similar to LA (1.5 × 102 CFU/g). Similarly LGG also colonized the large intestine in higher numbers than LA, with maximum colonization in the cecum (1.7 × 106 CFU/g). Unlike LA, no LGG was detected (detection limit, 10 CFU/g) in the extraintestinal tissues. On PBCD7 the LA colonization pattern remained similar to PBCD1, except more bacteria (>1-log) were recovered from all intestinal tissues and in higher numbers from all the extraintestinal tissues. However, LGG colonization was similar to PBCD1 and was also recovered from spleen (1 pig), liver (3 pigs), and MLNs (1 pig) on PBCD7. Translocation of both LA and LGG to secondary lymphoid organs (spleen, MLN) may serve for development of appropriate protective and controlled immune responses in the host.26,27 Rectal swab cultures indicated that both LA and LGG shedding increased from PBCD1 (LA, 2.2 × 104 CFU/g; LGG, 8 × 104 CFU/g) to PBCD7 (LA, 6 × 106 CFU/g; LGG, 6.5 × 105 CFU/g) consistent with the increased intestinal colonization. Ileum in early (PBCD1) and duodenum in later (PBCD7) stages are the major sites of transcriptome changes in response to probiotic colonization A summary of transcriptome responses following probiotic administration is listed in Table 1. Comparison of differentially expressed transcripts revealed more changes in the ileum for PBCD1 and in the duodenum for PBCD7, irrespective of probiotic bacteria treatment. On PBCD1 LA modulated 286 genes in ileum, whereas LGG modulated 292 genes. On PBCD7 in duodenum, LA affected 137 genes, and in contrast, LGG affected 569 genes. Another trend noted for both lactobacilli strains at both times was that more genes were downregulated in duodenum and upregulated in ileum, with the exception of ileum for LGG at PBCD 7. The transcriptional changes observed by microarray analyses were further confirmed by RT-qPCR for selected genes (Fig. S1), which also indicated a similar overall trend in the transcription. The microarray-generated transcripts were further mapped to available gene annotations in the IPA. Only up to 55% of transcripts were mapped due to lack of complete pig genome annotation. The number of IPA-mapped genes was further compared across probiotic strains, tissues, and days

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tested for the prevention or treatment of various pathological conditions. Numerous clinical studies have shown the beneficial role of Lactobacillus rhamnosus GG (LGG) in treatment of acute diarrhea in pediatric patients.8-10 LGG was effective in decreasing both duration and frequency of rotaviral diarrhea and also antibiotic-associated diarrhea in neonates.11-13 People consuming L. acidophilus (LA) NCFM had reduced fever, cough, and runny nose.14 Additionally, LA strains have also been shown to possess anti-inflammatory and breast cancer inhibition effects.14 These studies demonstrate that probiotics mediate their effects in a species and strain specific manner; however, mechanisms are still largely undefined.15 The Lactobacillus strains exert their beneficial effects in multiple ways: producing antimicrobial substances, enhancing barrier functions (i.e., improved gut integrity), producing cryoprotective heat shock protein (Hsp72) and antiapoptotic proteins (p40 and p75), regulating Th1-Th2 balance, enhancing B cell and IFN responses, and inhibiting cytokine induced apoptosis.16-22 Additionally, the type and timing of colonization of Lactobacillus strains influence the early neonatal gut mucosal immune responses and have a significant impact on resistance to diseases postnatally; however, further studies are required to understand the underlying molecular mechanisms. Investigations into these basic mechanisms will identify specific molecules and pathways mediating the beneficial effects on the host, which in turn aid in probiotic strain selection to modulate the specific clinical conditions. Investigation of in vivo probiotic-host cell interactions is difficult considering the complex gut microflora in infants. Germ-free animal models are critical to dissect the molecular interactions of probiotics. The neonatal piglets are similar to infants in terms of anatomy, physiology, and mucosal immunology and serve as a useful model to elucidate mechanisms of probiotic modulation of neonatal host responses.23 In the present study, we used neonatal Gnotobiotic (Gn) pig model to better understand L. rhamnosus (LGG) and L. acidophilus (LA) strain-specific responses. In vivo strain-specific colonization dynamics were investigated by conventional bacterial culturing and host-microbe interactions by in vivo transcriptome analysis of the small intestine (SI). We used duodenum and ileum for transcriptome analyses because they represent both inductive (ileum) and effector (duodenum) mucosal sites to assess probiotic-host interactions. The duodenum is the first segment of the SI to come in contact with probiotic bacteria, and it also helps in minimizing adaptive changes that probiotics might go through during passage of the intestinal tract.24 The ileum contains specialized structures (Peyer patches) for sampling and induction of immune responses to antigens and is also the site of enteropathogenic bacterial invasion.25 We investigated both early (post-bacterial colonization day 1, PBCD1) and later (PBCD7) transcriptome responses to probiotics. Comprehensive analysis of our microarray data indicated that both LA and LGG modulated expression of genes regulating host metabolism, mucosal cell integrity, and immunity in neonatal Gn pigs. Immune modulation and carbohydrate metabolism genes were most affected in LGG, whereas energy and lipid metabolism

Table 1. Summary of gut transcriptome responses to LA and LGG* PBCD

Tissue

All genes*

Up-regulated

Down-regulated

LA

1

Duodenum

157

73

84

LA

1

Ileum

286

161

125

LGG

1

Duodenum

183

66

117

LGG

1

Ileum

292

189

109

LA

7

Duodenum

137

58

79

LA

7

Ileum

108

74

34

LGG

7

Duodenum

569

259

310

LGG

7

Ileum

145

64

81

*The numbers represent the total number of transcripts that are differentially expressed in response to probiotic inoculation compared with uninoculated controls. The up-regulated and down-regulated genes were determined using a cutoff ± ≥ 2 or ≤ 2, respectively, compared with the uninoculated control group. PBCD refers to post bacterial colonization day.

post-colonization. On PBCD1 numbers of mapped genes were not significantly different across strains and tissues; however, significant differences were observed in duodenum on PBCD7 where LGG modulated higher numbers (2.1-fold) of unique as well common mapped genes compared to LA (Fig. S2; Table S1). Cellular growth and proliferation, inflammatory response, and immune cell trafficking pathways were modulated by LA and LGG To determine the relationship of genes identified in our microarray analysis using IPA, we compared the major welldefined molecular (canonical) pathways that were significantly affected by probiotic administration. Our results highlighted the distinct modulation of host gene expression in duodenum and ileum by both LA and LGG (Fig. 1A and B). Specifically, in most categories, LGG induced a pronounced response in duodenum, particularly on PBCD7, whereas an opposite trend was observed with LA, which induced a pronounced response on several pathways in ileum on PBCD7. On PBCD1 in duodenum, LGG modulated pathways associated with inflammatory response, hematological system development, and immune cell trafficking; however, both LA and LGG modulated cell-to-cell signaling and interaction, cellular growth, and proliferation pathways. On PBCD7 LGG also modulated cellular growth and proliferation, inflammatory response, and immune cell trafficking, whereas both LA and LGG affected cell signaling and cellular growth and proliferation pathways. Contrastingly, on PBCD7 in ileum, LA-modulated host tissue development, cellular movement, immune cell trafficking, and cell-to-cell signaling pathways, while both LA and LGG modulated inflammatory response and free radical scavenging pathways. Collectively, our analyses highlight the unique and common major cellular pathways modulated in duodenum and ileum by LA and LGG. In the early stage at PBCD1, LA and LGG administration affected cell morphology, cellular movements, and antiinflammatory genes in ileum To better understand the biological relevance of transcriptome responses observed after probiotic colonization, we analyzed the networks resulting from these transcriptome responses using IPA. The top affected networks are shown in Table 2. The top network in each category was compared (LA vs. LGG)

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and further analyzed using “Molecular Activity Predictor” (MAP). The predicted activities of molecules were in turn linked to significant canonical pathways representing the major underlying biological processes. The gene regulatory networks with allied canonical pathways are depicted (Fig. 2; Figs. S3 and S4). On PBCD1 the top network associated with LA in ileum was cellular movement, hematological system development and function, and immune cell trafficking. This network was linked to the canonical pathway involved in glucocorticoid receptor (GR) signaling. The majority of molecules involved (A2M, Akt, ERK, ERK1/2, GPCR, IL1, JNK, MAP2K1/2, MAPK, Mmp, NFκB [complex], P38 MAPK, PI3K [complex], Pka, and TNF [family]) were predicted to be inhibited (Fig. 2). MAPK, ERK1/2, and JNK have an important function in dynamic regulation of the cell cytoskeleton, tight junction (TJ), and epithelial barrier function.28 Selective interaction of cytoskeleton protein networks provides a structural framework for cell morphology, stabilizes the other membrane systems, and mediates cellular movements.29 This suggests that LA modulates the host cell morphology and cellular movement processes by selectively inhibiting these proteins. Further, glucocorticoids act through GR signaling and thereby mediate anti-inflammatory, anti-proliferative, and immunomodulatory activity.30 Dampening of GR signaling by LA indicated probable early anti-inflammatory and immune modulatory responses in the ileum, permitting more effective colonization with higher CFU observed vs. duodenum. Similarly, the top network associated with LGG in ileum was cell morphology, cellular function and maintenance, and cell cycle, which was also linked to the GR signaling canonical pathway (Cbp/p300, ERK1/2, Histone h3, HMGB1, IFNβ, MAPK, MEK, P38 MAPK, and TGFβ). The majority of the molecules involved in this pathway were predicted to be inhibited (Fig. 2). Cbp/p300 is a transcriptional co-activator protein that, besides exerting tumor-suppressive effects, also modulates proliferation, differentiation, and apoptosis in various cell types.31 It is likely that inhibition of Cbp/p300 by LGG stimulates cell proliferation, differentiation, and anti-apoptosis. LGG also seems to mediate host anti-inflammatory effects like LA by modulating genes associated with GR signaling in ileum.

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Group

In the later stage at PBCD7, LA and LGG modulated cell signaling and metabolism genes in duodenum On PBCD7 the top network associated with LA in duodenum was cell-to-cell signaling and interaction, tissue development, cell death, and survival. This network was associated with canonical pathway involved in integrin linked kinase (ILK) signaling (CDH1, ERK1/2, FOS, JUN, and VIM). The associated molecules were predicted to be inhibited (Fig. 3). The ILK signaling connects integrins to the cytoskeleton, thereby mediating cell signaling and also has important roles in cancer progression, and is a valid therapeutic target in cancer research.32,33 Downregulation of ILK signaling is likely to favor more colonization (>1 × 105) vs. PBCD1. The inhibitory status of transcriptional factors such as FOS and JUN, which downregulates the release of inflammatory cytokine (anti-inflammatory effects),34 is likely to decrease innate immunity to promote LA colonization. The top network associated with LGG in duodenum was cell cycle, carbohydrate metabolism, cellular function, and

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maintenance, which was linked to the canonical pathway involved in thyroid receptor complex (TR/RXR) activation signaling (Fig. 3). The corresponding molecules (Akt, G6PC, mTORC1, mTORC2, N-Cor, NCOA3, PEPCK, Rxr, thyroid hormone receptor, and TSH) were predicted to be activated except TSH (Fig. 3). Activation of TR/RXR signaling affects various processes including lipid/carbohydrate/steroid metabolism, thermogenesis, and CNS function.35 In addition, functional Akt and mTORC1 complex promotes lipid anabolism in the liver,36 and activation of G6PC (glucose-6-phosphatase), has consequences for host carbohydrate metabolism, as it is a key enzyme in glucose homeostasis37 (Table 3; Table S2). The genes associated with metabolism, cellular integrity, and immune responses were either commonly or uniquely modulated by LA and LGG Probiotic strain-specific host responses are well documented.22,25 The growth-phase-specific role for probiotic strains has also been reported recently.24 In this regard, our data

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Figure 1. Major cellular pathways (canonical pathways) modulated by LA and LGG were generated using ingenuity biological process analysis. These pathways were compared in duodenum and ileum for both days. The selected pathways were the significantly modulated ones and were above the threshold (-log [p value)] analyzed using IPA. Numbers on the top of the bars represent the total number of genes involved in a given canonical pathway. The distinction between probiotic strains was based on log p value and number of genes involved. (A) duodenum and (B) ileum.

Table 2. Top gene regulatory networks associated with LA and LGG in duodenum and ileum based on focus molecules and scores* LA Score

Top network involved

Day 1 Duodenum

Score

Focus molecules

45

20

Day 1 Duodenum

(1) Free Radical Scavenging, Small Molecule Biochemistry, Cell- To-Cell Signaling and Interaction

52

23

(1) Cell-To-Cell Signaling and Interaction, Hematological System Development and Function, Immune Cell Trafficking

(2) Protein Synthesis, Renal and Urological System Development and Function, Developmental Disorder

29

14

(2) Cancer, Cell-To-Cell Signaling and Interaction, Tumor Morphology

35

16

(3) Immunological Disease, Organismal Injury and Abnormalities, Cell-To-Cell Signaling and Interaction

22

11

Day 1 Ileum

Day 1 Ileum

(1) Cellular Movement, Hematological System Development and Function, Immune Cell Trafficking

35

16

(1) Cell Morphology, Cellular Function and Maintenance, Cell Cycle

46

19

(2) Neurological Disease, Molecular Transport, Cellular Development

33

15

(2) Developmental Disorder, Hereditary Disorder, Metabolic Disease

26

13

(3) Cancer, Reproductive System Disease, Cellular Development

22

11

Day 7 Duodenum

Day 7 Duodenum

(1) Cell-To-Cell Signaling and Interaction, Tissue Development, Cell Death and Survival

46

21

(1) Cell Cycle, Carbohydrate Metabolism, Cellular Function and Maintenance

27

16

(2) Carbohydrate Metabolism, Lipid Metabolism, Small Molecule Biochemistry

35

18

(2) Developmental Disorder, Hereditary Disorder, Neurological Disease

27

16

(3) Cell-To-Cell Signaling and Interaction, Cellular Assembly and Organization, Cellular Function and Maintenance

29

15

(3) Hereditary Disorder, Organismal Injury and Abnormalities, Reproductive System Disease

27

16

(4) Cell Signaling, Lipid Metabolism, Small Molecule Biochemistry

29

15

(4) Lipid Metabolism, Small Molecule Biochemistry, Molecular Transport

25

15

(5) Lipid Metabolism, Molecular Transport, Small Molecule Biochemistry

24

15

Day 7 Ileum

Day 7 Ileum

(1) Cellular Assembly and Organization, Skeletal and Muscular System Development and Function, Tissue Development

38

16

(1) Free Radical Scavenging, Hereditary Disorder, Ophthalmic Disease

41

17

(2) Cellular Movement, Renal and Urological System Development and Function, Cardiovascular System Development and Function

32

14

(2) Cardiovascular System Development and Function, Tissue Morphology, Cellular Development

26

12

*Only networks with focus molecules 10 or higher are shown, where “score” reflects number of network eligible molecules; the higher scores indicate that the given network is more likely modulated by probiotic treatment; Focus molecules’ are genes and molecules that are affected by the probiotic treatment and are considered for generating networks.

showed differentially expressed genes in different functional categories, particularly those involved in metabolism (Table 3; Table S2). Both LA and LGG modulated the genes involved in energy metabolism, whereas LA and LGG negatively regulated cytochrome subunits’ genes (e.g., COX1) in duodenum and positively regulated ATPase subunits (e.g., ATP5F1) in ileum. Similarly, LGG downregulated the pyruvate kinase isozymes and phosphoglycerate mutase 2 (PGAM2) genes involved in glycolysis, but LA upregulated the gluconeogenesis gene

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phosphoenolpyruvate carboxykinase (PCK1). Conversely, in protein metabolism LA downregulated the 3-oxoacid CoA transferase 1 gene (OXCT1) involved in branched chain amino acid synthesis, whereas LGG upregulated the branched chain amino-acid transaminase 2 mitochondrial (BCAT2) gene also involved in branched chain amino acid synthesis. Genes involved in dietary fat metabolism (PNLIP) and cholesterol clearance (APOA1) were upregulated by both LA and LGG. LA and LGG also modulated genes involved in vitamin and mineral

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Top network involved

LGG Focus molecules

metabolism. A majority of genes were induced for vitamin A (e.g., RDH) and vitamin D (e.g., CYP24A1) metabolism. LA also induced the gene responsible for iron storage (FTH1). Our data also supports common pathway modulation in duodenum and ileum: for example, the tight junction signaling on day 1, although the mechanistic regulation may vary for both LA and LGG due to involvement of different up stream regulators (Table S3). In duodenum transcription of catenin, claudin, and FOS molecules were affected where catenin was downregulated and claudin and FOS molecules were upregulated. In ileum, however, catenin, actin (α, αl and α2), FOS, and myosin (heavy and light chain) were involved, and all molecules were predicted to be activated except NF-κB. The NF-κB signaling inhibits MLCK (myosine light chain kinase) to decrease the permeability of tight junctions in the ileum (Fig. S5A and B), and this may suggest that MLCK inhibition may limit the extraintestinal dissemination of LGG to spleen, liver, and MLN on PBCD1. Both LA and LGG induced genes involved in cellular integrity such as sialomucin (CD164 molecule), integrin, β 3 (ITGB3), heat shock 27 kDa protein 8 (Hsp27), and glucagonlike peptide-2 receptor (GLP2R) (Fig. 4; Table S4). Additionally, both LA and LGG tended to downregulate the innate immune response genes in the early stages (PBCD1) to favor colonization.

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For example, cytosolic- and membrane-bound forms of glutathione S transferase (GSTA1), lysozyme (LYZ), polymeric immunoglobulin receptors (PIGR), surfactant protein D (SFTPD), and an antibacterial protein regenerating islet-derived 3 gamma (REG3G) were downregulated in duodenum (Fig. 4; Table S4). However, in the later stages (PBCD7), complement component C9 and C8G, cellular innate mediators, CD1d, nonclassical MHC I, and Th1-specific IFN-induced chemokine CXCL9, were induced by both LA and LGG.

Discussion The beneficial role of probiotics in nutritional physiology is less documented compared with its anti-diarrheal effects and role in immune stimulation.38 However, few studies highlighted the beneficial role of lactobacilli in enhancing mineral and nutrient absorption, vitamin metabolism, or modulation of intestinal physiology.39 Human interventional studies in children using L. rhamnosus and L. acidophilus showed improvement in weight and size of the subjects, likely due to an increased feed conversion ratio; 40,41 however, mechanisms still remain unclear.39 Our study provides mechanistic insights into the role of both LA and LGG in nutrient metabolism. LA upregulated two important host genes, ferritin (FTH1) and ceruloplasmin (Cp), which are involved in

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Figure 2. Top associated networks with their linked canonical pathways (CP) in ileum on PBCD1. In the graphical representation of a network, genes or gene products are represented as nodes, and the biological relationship between two nodes is represented as an edge (line). Human, mouse, and rat orthologs of a gene are represented as a single node in the network. The intensity of the node color indicates the degree of up- (red) or down- (green) regulation, and more confidence predicted activation (blue) or inhibition (brown) of a given gene. Edges are displayed with various labels that describe the nature of the relationship between the nodes. Both LA and LGG were associated with glucocorticoid receptor (GR) signaling canonical pathway. The connecting sky blue line indicates the associated molecules with their predicted biological status, whereas the pink lines highlight the direct relation (solid line) between the genes. Only genes that are involved in canonical pathway are shown, and indirect interactions are depicted in dashed arrows with different colors representing the predicted biological status as indicated in the legend.

iron and copper homeostasis.42 Furthermore, upregulation of antimicrobial peptide hepcidin (HAMP) and ferritin, the host iron sequestration genes, limit iron availability to intruding pathogens, thereby preventing pathogen establishment in the host.43 Both LA and LGG may also play a role in bioavailability of small molecules, calcium, and phosphorus. Host intestinal absorption and homeostasis of calcium and phosphorus requires the vitamins A and D. The gene product of CYP2D25 and CYP24A1 encodes vitamin D 25-hydroxylase and 1, 25-dihydroxyvitamin D3 24-hydroxylase, respectively, the key enzymes in the vitamin D metabolism. Our data support the strain specific induction of CYP2D25 gene by LGG in duodenum on PBCD7 and common induction of CYP24A1 gene on PBCD1. It is interesting to note that LGG on PBCD1 differentially modulated the CYP24A1 gene in the duodenum and ileum, suggesting calcium absorption was favored in duodenum (Table 3; Table S2). Likewise, β-carotene 15,15′-monooxygenase (BCMO1) gene involved in vitamin A metabolism was upregulated by LA in duodenum (PBCD1); conversely, LGG in both tissues upregulated RDH16, retinol dehydrogenase 1, an enzyme involved in the visual cycle (Table 3; Table S2). Both vitamins A and D can modulate the innate and adaptive immune responses as well as metabolites of vitamins A and D can induce tissue-specific immune responses and have been investigated for preventing and/or treating inflammation and autoimmunity.44 Previously, colonization of B. thetaiotaomicron in germ-free mice produced changes in host genes involved in breakdown and

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absorption of complex carbohydrates and lipids.45 In our study lactobacilli administration altered expression of genes involved in glycolysis and/or gluconeogenesis. For example, pyruvate kinase isozymes R/L, like LOC100621940 and phosphoglycerate mutase 2 (PGAM2), were negatively regulated, whereas phosphoenolpyruvate carboxykinase 1 (PCK1) and glucose-6phosphatase, catalytic subunit (G6PC) were positively regulated. This suggests a potential role for lactobacillus in host carbohydrate metabolism (Table 3). Lactobacillus contributes to host lipid metabolism through their ability to metabolize bile acids, the primary function of which is to emulsify fats. Hence, probiotics seem to regulate how much fat the body can absorb.28 LA on PBCD1 in duodenum induced genes pancreatic lipase-related protein 1 (PNLIPRP1) (involved in breakdown of triacylglycerols) and pancreatic colipase (CLPS) (prevents the inhibitory effect of bile salts on lipase), whereas, on PBCD7 in duodenum, LGG upregulated pancreatic lipase (PNLIP), the primary enzyme required for hydrolyzing dietary fat. Recently, it was shown that yogurt containing probiotic bacterial strains, L. acidophilus and B. lactis, had a cholesterol-lowering effect in hypercholesterolemic subjects.46 Both LA and LGG on PBCD7 in duodenum induced a 300-fold upregulation of apolipoprotein A-I (APOA1), a major protein component of high density lipoprotein (HDL) in plasma, promoting cholesterol efflux from tissues to the liver for excretion and helping to clear cholesterol from arteries.47 L. casei Shirota and B. breve48 have been shown to enhance colonic protein and ammonia metabolism in healthy humans. In our study genes

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Figure 3. Top associated networks with their linked canonical pathways in duodenum on PBCD7. LA was associated with integrin linked kinase (ILK) and LGG with thyroid receptor complex (TR/RXR) activation canonical pathways. The connecting sky blue line indicates the associated molecules with their predicted biological status, whereas the pink lines highlight the direct relation between the genes. Only genes that are involved in canonical pathway are shown, and indirect interactions are depicted in dashed arrows with different colors representing the predicted biological status as indicated in the legend.

Table 3. Metabolism genes influenced by administration of probiotics LA and LGG in duodenum and ileum.* Gene symbol and functions

PBCD1 LA

Small molecules metabolism

Duo

Ile

PBCD1 LGG Duo

Ile

­↑

↓

PBCD 7 LA Duo

Ile

PBCD7 LGG Duo

Ile

(1) Vitamin and mineral BCMO1, Vitamin A metabolism

­↑

CYP24A1, Vitamin D metabolism

­↑ ↑­

DIO3, Thyroid hormone metabolism

­↑

RDH16, Vitamin A metabolism

­↑

­↑

FTH1, Iron storage

­↑

HAMP, Macrophage iron storage

­↑

CP, Copper and iron homeostasis

­↑ ­↑

CYP2D25, Vitamin D metabolism (2) Drug metabolism TPMT, Metabolizes thiopurine drugs

↓

DPEP1, Renal metabolism of glutathione and conjugates

↓

↓ ­↑

FMO1, Metabolism of drugs and xenobiotics

↓

DHRS7, Metabolism of steroid, prostaglandins, retinoids, lipids and xenobiotics Macro molecules metabolism (1) Carbohydrate ­↑

PCK1, Gluconeogenesis

­↑

GPD1, Link between carbohydrate and lipid metabolism PDK4, Inactivate pyruvate dehydrogenase

­↑­

G6PC, Key enzyme in glucose homeostasis

­↑

AMY2, Hydrolyze oligo and polysaccharides

­↑

LOC100621940, Plays a key role in glycolysis

↓ ↓

PGAM2, Involved in glycolysis (2) Lipid metabolism CLPS, prevent the inhibitory effect of bile salt on lipase

­↑

PNLIPRP1, Role in dietary fat digestion

­↑ ­↑­

APOA1, Role in cholesterol metabolism

­↑­

PNLIP, Primary enzyme in hydrolyzing dietary fat

­↑

LIPE, Hydrolyze the first fatty acid from a triacylglycerol

­↑

(3) Protein metabolism OXCT1, Branched chain aa degradation, synthesis and degradation of ketone bodies

↓

MTRR, Helps in process of aa synthesis

­↑ ­↑

OAZ2, ornithine decarboxylase inhibitor thereby prevent polyamine synthesis ↓

SADC, Amino acid (aa) metabolism and urea cycle

↑­

BCAT2, Synthesis of the branched chain aa

­↑

OAZ1, ornithine decarboxylase inhibitor,prevent polyamine synthesis

↓

HPD, Break down the aa tyrosine (4) Energy metabolism COX3, Cytochrome c oxidase subunit

↓

COX1, cytochrome c oxidase subunit I

↓

ATP5B, Mitochandrial ATPase synthase

↓

ATP5F1, Mitochandrial ATPase synthase

­↑

↓

↓

­↑

*Only selected genes in each category are shown with ‘↓’ and‘↑’ referring to down or upregulation, respectively. Comprehensive list of all the genes involved in metabolism with fold change values and description are listed in Table S2. The upregulated/downregulated genes were determined using a cut off ± ≥ 2 or ≤ 2, respectively compared with the uninoculated control group. www.landesbioscience.com

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CYP4A21, Vitamin D metabolism

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signaling is attributed in metabolic signaling pathways (glucose, fatty acid, and cholesterol metabolism) besides morphogenesis, cell proliferation, cell differentiation, and cell death.35 Alterations in the epithelial barrier functions are implicated in various intestinal disorders, and probiotics are suggested to enhance or maintain the epithelial barrier, but the molecules mediating these effects are only partially defined. In our study both LGG and LA altered gene expression in canonical pathways involving cell signaling, tissue development, cellular growth, and proliferation. For example, both LA and LGG ingestion enhanced expression of glucagon-like peptide-2 receptor (GLP2R) gene (Fig. 4). The GLP2R regulates intestinal growth stimulation and upregulation of villus height in the small intestine, concomitant with increased crypt cell proliferation and decreased enterocyte apoptosis. Enterocyte transcriptome profiling in neonatal mice has shown that ingestion of probiotic L. reuteri strains altered expression of genes associated with multiple canonical pathways involving cell motility, as well as increased enterocyte migration and proliferation in the ileum in a strain-specific manner.22 ILK signaling stabilizes enterocyte migration and has been implicated in intestinal epithelial cell proliferation regulation and certain bacteria hijack ILK signaling to stabilize focal adhesions and prevent cell detachment.49-51 It is possible that LA might exploit ILK signaling to colonize the Gn pig intestine (Fig. 3). LGG also induced expression of claudin-8, which is associated with decreased paracellular gut permeability52 Figure 4. Genes involved in cell integrity and immunity. The gene functions were determined (Table S3). Further, our results suggested an using IPA and NCBI database and were manually compared with their regulated status. The anti-apoptotic role for both LA and LGG on genes involved were color coded to indicate regulated status (grey, downregulation; black, the host cell through induction of heat shock upregulation). The genes responsible for innate immunity were commonly regulated in both proteins (e.g., Hsp27) (Fig. 4). Increased LA and LGG. expression of Hsp27 has been shown to suppress stress- and receptor-induced involved in catabolism of branched chain amino acids (3-oxoacid apoptotic pathways. Also, Hsp27 increases the antioxidant CoA transferase 1 OXCT1), tyrosine (4-hydroxyphenylpyruvate defense of cells by decreasing intracellular reactive oxygen species dioxygenase HPD), and arginine (arginine decarboxylase (ROS).53 Thus, both LA and LGG may contribute to maintaining SADC) were negatively regulated, whereas genes involved in gut homeostasis; however, LGG may play a significant role as a amino acid synthesis (5-methyltetrahydrofolate-homocysteine probiotic in influencing gut epithelial integrity. methyltransferase reductase, MTRR) and branched chain amino Our study also highlighted the immunomodulatory acid synthesis (branched chain amino-acid transaminase 2, functions of LA and LGG. Both LA and LGG on PBCD1 mitochondrial BCAT2) were positively regulated (Table 3). This showed downregulation of various genes related to innate suggests that both LA and LGG may play a role in host protein immune responses, such as anti-microbial peptides (REG3G, metabolism, and this may have been mediated through activation surfactant protein D), lysozyme, antigen presentation (MHC of TR/RXR signaling (Fig. 3), as in the case of LGG. TR/RXR class 1 and 2) and regulation (CD32), and immune cell

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Understanding these interactions will provide information for the rational treatment of disease phenotypes with known probiotics and will have significant implications in personalized healthcare medicine.

Materials and Methods Bacterial strains and growth conditions The Lactobacillus rhamnosus GG (ATCC 53103) and L. acidophilus NCFM™ (ATCC 700396) strains were routinely cultured in Lactobacilli MRS media (Hardy Diagnostics) at 37 °C with BD GasPak EZ anaerobic container system (Becton, Dickinson and Company). The freezer stocks of strains were stored at -80 °C in MRS medium with 30% glycerol (v/v). The cells from the freezer stock were plated on MRS agar, and overnight-grown culture was used to inoculate 5 ml of MRS broth and grown overnight in anaerobic conditions. The OD600nm was measured to calculate the colonization dose of 1 × 108 CFU per pig. Ethical statement and animal experiment All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of The Ohio State University. Piglets were derived surgically from nearterm sows (Landrace × Yorkshire × Duroc) and maintained in sterile isolators as previously described.59 Conventional culture methods (blood agar plates and thioglycolate broth culturing) were used to confirm the sterility of Gn pigs before probiotic administration. Gn pigs were divided randomly into three groups: group 1, LGG (n = 8); group 2, LA (n = 8); and group 3, noncolonized controls (n = 6). The pigs were inoculated orally with 1.0 × 108 CFU per pig at 3 d of age. Throughout the experiment, animals received the same type of feed (ultra-high temperature processed commercial cow milk [Parmalat]). The animals were euthanized on PBCD1 and 7, and tissue samples (duodenum, jejunum, ileum, cecum, colon, rectum, spleen, liver, and mesenteric lymph nodes [MLN]) were collected. In addition, rectal swabs were also collected on PBCD1, 3, and 7 to monitor colonization dynamics. Tissue samples were suspended in buffered peptone water, homogenized, suspensions were 10-fold serially diluted, and then 100 μL of each dilution was spread onto lactobacilli MRS agar plates and incubated for 24 h. The CFU/g of tissue or per ml of rectal swab was determined. The statistical analysis was performed using independent group t test or paired t test to compare colonization between strains, tissues, and days. The P value of ⩽0.05 was considered as significant. RNA isolation and analysis We investigated both early (PBCD1) and late (PBCD7) transcriptome responses to probiotics in the duodenum and ileum. Since probiotic bacteria begin to colonize by day 1 and attain the maximum colonization by day 7 in these pigs, we selected these time points for analysis. A modified Trizol method was followed for RNA extraction. Briefly, the tissue samples were homogenized with Trizol reagents (Life Technologies) followed by chloroform treatment. Subsequently, the samples were centrifuged at 12 000 × g to obtain a clear lysate. The lysates were mixed immediately with an equal volume of 70% ethanol (v/v),

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trafficking (CCL20/28, only LGG; Fig. 4; Table S4), which may allow early colonization and adaptation of these probiotics to the host. GR signaling leads to enhanced release of proinflammatory cytokines IL-1β, TNF-α, and IL-6,54 hence, it may be conceivable that downregulation of GR signaling at an early stage by both LA and LGG could suppress the initial host response that otherwise prevents probiotic colonization (Fig. 2). Once colonized on PBCD7, both probiotics induced stimulatory effects in duodenum and ileum, as indicated by higher expression of soluble (complement component 9 and 8G genes) and cellular innate mediators (non-classical MHC class I CD1d expressed on NKT cells). Furthermore, both LA and LGG induced CCL9 expression, which is a Th1-specific IFN induced chemokine similar to CXCL10 and CXCL11 genes,55 and LA also increased granzyme A/B, which is expressed by cytotoxic T cells and NK cells (both associated with Th1 responses), suggesting that these probiotics may favor Th1 immune responses. Interestingly, in our previous studies we have shown Th1 immunomodulating effects of LA and LGG (in combination with Bb12) on attenuated HRV vaccine in a Gn pig model,3,20 suggesting that the initial Th1 microenvironment (observed on PBCD7) induced by these probiotics may result in Th1-biased adaptive immune response to specific microbial agents. Thus both LA and LGG may be useful as adjuvant for viral vaccines and infections, where Th1 immune responses are critical. Though our study used a Gn pig model, similar observations have been made using other conventional animal models, as well as in clinical trials, supporting the usefulness of Gn pigs in elucidating mechanisms underlying the beneficial roles of probiotics. For example, comparison of transcriptome responses of germ-free piglets vs. the piglets with intestinal microbiota indicated that genes involved in biological processes like epithelial cell turnover, nutrient transport and metabolism, xenobiotic metabolism, JAK-STAT signaling pathway, and immune response were altered.56 Similarly, administration of LA and LGG in a human interventional study identified mucosal transcriptional responses associated with immune response, tissue growth and development, and ion homeostasis.18 In our study, genes involved in nutrient transport and metabolism, immune responses, and epithelial cell turnover are also the major classes of genes that were differentially regulated by the colonization of Gn pigs with lactobacillus species. In addition, previous studies using Gn and conventionalized mice revealed major molecular responses in metabolism, intestinal morphology and cell proliferation, and immunity as early as days 1–4 postconventionalization, with pronounced changes occurring after day 4 post-conventionalization.57,58 Consistent with these findings, in our study more robust responses were observed on PBCD7, particularly in duodenum, illustrating the tissue-specific changes in the biological processes. However, detailed temporal analysis of transcriptome responses to lactobacilli colonization in neonatal Gn and conventional pigs (mimicking the infants) would provide better understanding of the biological processes modulated by these known probiotics. In addition, studies of the host proteome and metabolome should further enhance our understanding of how probiotic bacteria confer health benefits.

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ileum by LA and LGG. The significant (± 2 ⩾ or ⩽ 2) regulated genes were uploaded into IPA along with the gene identifiers and corresponding fold change values. Each gene identifier was mapped to its corresponding gene object in the Ingenuity Pathways Knowledge Base (IPKB), and a set of relevant networks, focus genes, and canonical pathways were identified. The significance of the association between the genes from the data set and the canonical pathways was measured as described in IPA. For network analysis, networks of differentially expressed genes were algorithmically generated based on their connectivity. Two genes are considered to be connected if there is a path in the network between them, and the highly interconnected networks likely represent significant biological function. The functional analysis of a network identifies the biological functions and/or diseases that were most significant to the genes in the network, and the functional analysis of the entire data set identifies the biological functions and/or diseases that are most significant to the data set. Fischer’s exact test was used to calculate a P value for each biological function assigned to that network. Reverse-transcription quantitative PCR (RT-qPCR) To validate the microarray data we tested the expression profiles of selected genes using RT-qPCR. The available similar sequences were searched using Tentative Consensus number (TC #) with Sus scrofa deposited reference sequence at http://compbio. dfci.harvard.edu/cgi-bin/tgi/gimain.pl?gudb=pig to design primers for RT-qPCR. Primers were designed and commercially synthesized using Integrated DNA Technologies (IDT). The primers used with their gene description are listed (Table S5). The cDNA was synthesized from total RNA using SuperScript® III First-Strand Synthesis SuperMix kit (Invitrogen). cDNA concentration was normalized, and RT-qPCR was performed using SensiMixPlus® SYBR RT-PCR Kit (Bioline) in a realplex 2 mastercycler (Eppendorf). The difference in expression of genes was calculated using the comparative threshold cycle (ΔΔCt) method63 to yield fold-difference in transcript levels. Disclosure of Potential Conflicts of Interest

No potential conflict of interest was disclosed. Acknowledgments

We gratefully acknowledge the technical assistance of Dr Juliette Hanson, Rich McCormick, Lindsey Good, Joshua Amimo, Isaac Kashoma, and Kyle T Scheuer. This work was supported by grants from the NIH, NCCAM R21AT004716 and NIAID R01 A1099451 (L.J.S. [PI], A.N.V., and G.R. [coPI]), and federal funds appropriated to the Ohio Agricultural Research and Development Center (OARDC) of The Ohio State University. Supplemental Materials

Supplemental materials may be found here: www.landesbioscience.com/journals/gutmicrobes/article/27877

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and RNA was extracted using RNeasy mini kit (Qiagen Inc.) followed by DNase treatment. The concentration and purity of RNA samples were determined using Nanodrop ND2000c spectrophotometer (Thermo Scientific) and agarose gel electrophoresis. RNA integrity and purity was further confirmed using Agilent 2100 bioanalyzer (Agilent Technologies). The samples with ratio of 18S:28S (Sus scrofa rRNA subunits) closer to 2 were used for microarray analyses. Transcriptome analysis The expression analysis was performed using Sus scrofa Sureprint 4 × 44k arrays (Agilent Technologies). The Agilent Sureprint 4 × 44k high-density arrays contain in situ synthesized 60 mer oligo’s representing 44  000 pig expressed sequence tags. Microarray hybridizations were performed according to the manufacturer’s instructions at the Biomedical Genomics Core facility, The Research Institute at Nationwide Children’s Hospital, Columbus, Ohio. In brief, samples were labeled with Cy3, purified using Qiagen columns, and checked for labeling efficiency using the Nanodrop. The labeled test and control samples were fragmented and hybridized to the array overnight. Microarray slides were then washed and scanned with Agilent G2505C Microarray Scanner, at 2 µM resolution. Subsequently, images were analyzed with Feature Extraction 10.10 (Agilent Technologies). Median foreground intensities were obtained for each spot. The data set was filtered to remove positive control elements and any elements that had been flagged as bad. Statistical analysis For the microarray intensity data, the data was background corrected and normalized with a quantile normalization method.60,61 A filtering method was applied to filter out low expression genes if more than 80% of arrays had expression level at or below the noise cutoff. Linear mixed effects’ models were used to account for the dependencies among observations from the same subject (each subject provided both ileum and duodenum samples). Treatment types, organ types, treatment days, and interactions among them were included as fixed effects in the model. A time factor was also included to control for the batch effects because the microarray samples were run on two different days from samples obtained from 2 independent experiments. For each comparison type I error was controlled by allowing for number of false positives among the tested genes.62 Because of the small sample size, to determine the expression of a gene is significantly different between conditions, we controlled type I error at 0.0003 by controlling 10 false positives out of the tested genes (around 30 000) for each comparison (cutoff for p-value = 0.0003). Again, due to the nature of the study, for the pathway analysis, we also included some genes with more than 2-fold changes, but with p-values larger than the cutoff. Ingenuity pathway analysis (IPA) The web-based pathway analysis tool, IPA (www.ingenuity. com, Ingenuity Systems®) was used to identify biological functions and molecular networks modulated in duodenum and

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