Exposure to Probiotic Lactobacillus acidophilus L-92 Modulates Gene Expression Profiles of Epithelial Caco-2 Cells

Abstract

To understand host gastrointestinal response after exposure to probiotic Lactobacillus acidophilus L-92, microarray analysis of cultured epithelial Caco-2 cells was performed. Of the 187 genes down-regulated after 4 h treatment with L-92, 25 were involved in RNA splicing; 12, in cell cycle; 8 were transcriptional regulators; 2 were involved in ubiquitin proteolysis; 2, in adhesion; 2, in meiosis; 2, in splicing; and 2 encoding cytokines. In the RNA splicing group, genes encoding small nuclear RNAs, nuclear pore complex interacting proteins, RNA binding motif proteins, and SMG1 homologs (phosphatidylinositol 3-kinase-related kinase) were identified. Among the only 13 genes up-regulated by the treatment, 5 were involved in histone structure, and 2 were involved in metabolism. Genes belonging to cell adhesion, transmembrane proteins, mitogen-activated protein kinase, immune response, DNA binding, inflammation, and protein synthesis groups were mainly up-regulated after 20 h of treatment, whereas no significantly down-regulated genes were observed. In the present transcriptome analysis, during the early stage of treatment (four hours of treatment) with L-92, genes involved in cell growth and cell meiosis were mainly repressed. During the late phase of treatment (20 h of treatment), the expression of the genes linked to cell adhesion activity and metabolism for cell growth was enhanced. From the present transcriptome analysis, we suggest that Caco-2 cells slow down cell death and turnover of RNA synthesis as an early response to L-92 treatment; at the late stage of treatment, the genes involved in cell proliferation, transcriptional activity, and apoptosis are activated.

Introduction

M any industrial probiotic bacterial strains have been developed that help manage a number of health issues, such as gastrointestinal (GI) disorders, inflammatory bowel diseases, allergic symptoms,^1
–3 and pathogenic infections. Among these probiotic strains, Lactobacillus acidophilus L-92 has been developed with anti-allergic effects for pollen allergy,⁴ perennial allergy,⁵ atopic dermatitis,^6,7 and the ability to control gastro-intestinal disorders.⁸ These results are based on clinical evidence from Japanese subjects. These health benefits, especially immunomodulatory effects, are thought to be caused by interactions between L-92 and the host, which are followed by secondary responses, mainly through the immune network. The adhesion of L-92 to epithelial cells of the GI tract may also be an important event for their communication with host GI cells and survival in the GI tract.

Recently, transcriptome analyses of human intestinal samples, collected from biopsies during clinical trials, were conducted to study host GI events.^9

–12 Studies have sought to understand the host response after the intake of probiotic cells. However, the collection of human samples during clinical trials is not an easy task; GI cells are strongly affected by diet, but it is difficult to control the subjects' diet.

Caco-2 cells are a well-established human epithelial cell line and have been used in many studies pertaining to transepithelial transport, metabolism, and disease.^13
–15 A recent study analyzed changes in the host response through the analysis of Caco-2 cells using microarray analysis.^16
–18 Caco-2 cells could be used to compare differences in probiotic characteristics of different bacterial strains. Of particular interest, Caco-2 cells overlayed on macrophage-like RAJI cells showed cross-communication in the transwell system.^19

–22 A transwell system using co-cultivating Caco-2 cells with macrophage-like cells, such as THP-1 cells and RAJI cells, serves as a useful tool for studying cross-talk between epithelial cells and immune cells.^23,24 In the use of the transwell system, inflammatory damage caused by pathogenic bacterial treatment and its rescue by some probiotics has been reported.²⁴

Based on a comparative proteome analysis using L. acidophilus L-92 and the less adhesive CP23 strain,²⁵ it was suggested that surface layer protein A (SlpA) of L. acidophilus L-92 could serve as an adhesion molecule with Caco-2 cells to aid the communication between L-92 cells and host cells. Results show L-92 to be more effective in releasing an inflammatory cytokine, IL-12, from splenocytes compared with CP23, which has less SlpA. Changes in some cytokines and chemokines after uptake of L-92 were reported in animal studies.^6,26 However, information processes, in addition to host metabolism, is required.

In the present study, using microarray analysis, we investigated changes in gene expressions link to metabolism occurring in GI epithelial cells after exposure to L-92 cells.

Materials And Methods

Bacterial strain

L. acidophilus L-92 strain was used from our stock culture collection and was maintained anaerobically at 37°C for 20 h. in Man Rogosa Sharpe (MRS) broth (Difco Laboratories, Detroit, MI).

Caco-2 cell culture

The human colonic cell line Caco-2 was obtained from the RIKEN Cell Bank (Tsukuba, Japan) and maintained in Dulbecco's modified Eagle's medium (Sigma, St. Louis, MO) containing 10% (V/V) of heat-inactivated fetal bovine serum (Hana-Nesco Bio, Tokyo, Japan), 1% (W/V) amino acids (Sigma), streptomycin (100 μg/mL), and penicillin (100 U/mL) (GIBCO, Grand Island, NY) in a 100 cm² dish (Corning Glass Works, Corning, NY) at 37°C in 5% CO₂/95% air. The culture medium was replaced every other day to maintain the cells. Monolayered cells were collected by adding 0.25% trypsin (Sigma) for 5 min, and were seeded at 4.5×10⁵ cells/well in 3.0 mL of the cell suspension.

L-92 treatment of Caco-2 cells

For the transcriptome analysis assay, Caco-2 cells were grown in six-well tissue plates (Asahi Glass Co., Ltd., Tokyo, Japan) at 37°C. The medium was changed every other day. The assay was performed on Caco-2 cells on day 21. On day 21, the L-92 cells cultured in MRS medium were collected by centrifugation at 13,000 g for 10 min, and resuspended in phosphate-buffered saline (PBS) (8.15 mM Na₂HPO₄12H₂O, 1.47 mM KH₂PO₄ 2.68 mM KCl, and 0.137 M NaCl, pH 7.4) containing 0.137 M NaCl. The L-92 cells were collected again by centrifugation at 13,000 g for 10 min. The washed cells were resuspended in PBS and heat treated at 100°C for 10 min. Then, 1×10¹⁰ cells of heat-killed L-92, suspended in 3 mL of culture buffer were added to each well of the culture plate. After centrifugation of the plate at 3000 g for 1 min to accelerate the association of L-92 and Caco-2 cells, the mixed cells were incubated at 37°C in 5% CO₂/95% air. After 4 and 20 h of incubation, the supernatant from the mixed cultured cells was discarded, and the Caco-2 cells were washed twice with PBS to remove nonbound cells. Finally, the Caco-2 cells were harvested using a cell scraper after the addition of cold PBS. After centrifugation, 1.5 mL of RNAlater solution (QIAGEN K. K., Tokyo, Japan) was quickly added to the collected cell pellet to extract RNA. The suspension was maintained at 4°C for 12 h, and then stored at −30°C. Total RNA was purified and used for cDNA synthesis after analysis of its quality. Untreated cells were used as controls. Three replicate samples were prepared for each group.

Microarray analysis

Total RNA was extracted using an RNeasy Mini Kit (QIAGEN, Valencia, CA). Double-stranded cDNA was synthesized from 5 μg of total RNA, and the cDNA was subjected to in vitro transcription in the presence of biotinylated nucleotide triphosphates. Human genome-wide gene expression was examined using the GeneChip^® Human Gene 1.0 ST Array (Affymetrix, Santa Clara, CA) containing oligonucoleotide probe sets for approximately 28,869 full-length genes and expressed sequence tags. The biotinylated cRNA was hybridized with the probe array for 16 h at 45°C. The hybridized products were stained with streptavidin-phycoerythrin, and then scanned with a Hewlett–Packard Gene Array Scanner (Palo Alto, CA). The fluorescence intensity of each probe was quantified using GeneChip Analysis Suite 5.0 software (Affymetrix). The level of gene expression was determined as the average difference using the GeneChip software.

Statistical and functional analysis of microarray data

Data analysis was performed using Genespring GX software, version 11.5.1 (Silicon Genetics, San Carlos, CA). Expression data were considered significant when they differed by at least 1.25 fold between the L-92 treatment and the PBS treatment, as P values of <.05 were observed when statistically analyzed by T test unpaired with Benjamini–Hochberg as post test.

Results

Changed gene expressions after four hours of treatment of L-92 cells

Most of the changes in gene expression in Caco-2 cells were relatively mild after treatment with L. acidophilus L-92. However, significant changes were observed. After the 4 h of treatment with L. acidophilus L-92, 187 genes were significantly down-regulated by <0.77-fold, and 12 genes were up-regulated by >1.25-fold in Caco-2 cells compared with nontreated cells, as illustrated in Figure 1. After 20 h of treatment, 45 genes were significantly up-regulated in Caco-2 cells by >1.25-fold (Fig. 1). For a detailed understanding of the response of the cultured Caco-2 cells, up-regulated and down-regulated genes were categorized based on the common pathways in KEGG analysis (www.genome.jp/kegg/pathway.html), as shown in Table 1. As illustrated in Figure 1, most of the characteristic changes in gene expression observed at four hours pertained to down-regulation. Of the genes down-regulated after the 4 h of treatment, 25 genes were classified as RNA splicing, 6 genes as transcriptional regulators, 2 genes as ubiquitin proteolytics, 2 genes as adhesion, 2 genes as meiosis, 2 genes as cytokines, and 2 genes as splicing (Fig. 1). In the RNA splicing group, genes encoding small nuclear RNAs, nuclear pore complex interacting proteins, RNA binding motif proteins, and SMG1 homologs (phosphatidylinositol 3-kinase-related kinase) were identified. After the 4 h of treatment, only five genes were involved in histone structure, two genes were involved in metabolism, and a further five other genes were up-regulated (Table 1).

FIG. 1.

Profile of the changed genes in the transcriptome analysis of Caco-2 cells after 4- and 20-h treatment with Lactobacillus acidophilus L-92. Genes categorized by KEGG analysis are shown in the square.

Table 1.

Changed Genes in Caco-2 Cells After Four Hours of Treatment of Lactobacillus acidophilus L-92

Category Gene description	Gene symbol	Genbank	Fold change	P value
Up-regulated genes
Nucleosome structure
Histone cluster 1, H2bf	HIST1H2BF	BC056264	1.40	0.003
Histone cluster 1, H2bh	HIST1H2BH	BC096116	1.34	0.005
Histone cluster 1, H1c	HIST1H1C	AK290947	1.30	0.006
Histone cluster 1, H4d	HIST1H4D	BC128104	1.31	0.001
Histone cluster 1, H1b	HIST1H1B	BC069101	1.31	0.010
Metabolism
Cytochrome P450, family 1, subfamily B, polypeptide 1	CYP1B1	U03688	3.51	0.000
TIMP metallopeptidase inhibitor 3	TIMP3		1.46	0.007
RNA synthesis
Polymerase (RNA) I polypeptide E, 53 kDa	POLR1E	BC001337	1.33	0.000
Phagosome
Coronin, actin binding protein, 2A	CORO2A	BC011690	1.46	0.000
MAPKK family
PDZ binding kinase	PBK	AF237709	1.34	0.000
Transmembrane
Phosphatidylinositol glycan anchor biosynthesis, class K	PIGK	AF022913	1.31	0.002
Ubiquitination
Ubiquitin associated and SH3 domain containing B	UBASH3B	BC007541	1.40	0.006
Down-regulated genes
RNA spliceosome, survailance
Small nucleolar RNA, H/ACA box 16A	SNORA16A	AK092096	−0.49	0.001
Small nucleolar RNA, C/D box 13 pseudogene 2	SNORD13P2	X58060	−0.58	0.002
Small nucleolar RNA, H/ACA box 38B (retrotransposed)	SNORA38B		−0.59	0.000
Small nucleolar RNA, H/ACA box 75	SNORA75		−0.61	0.002
Small nucleolar RNA, C/D box 15A	SNORD15A		−0.67	0.001
Small nucleolar RNA, H/ACA box 70	SNORA70	AK027197	−0.75	0.001
Small nucleolar RNA, H/ACA box 70B	SNORA70B		−0.76	0.004
Small nucleolar RNA, H/ACA box 61	SNORA61	AK092096	−0.76	0.042
Small nucleolar RNA, H/ACA box 51	SNORA51		−0.76	0.042
Small nucleolar RNA, H/ACA box 67	SNORA67	AK296664	−0.77	0.006
Nuclear pore complex interacting protein	NPIP	AK294177	−0.70	0.001
Nuclear pore complex interacting protein	NPIP	AF132984	−0.74	0.002
Nuclear pore complex interacting protein	NPIPL4	AK296338	−0.75	0.011
Nuclear pore complex interacting protein	NPIP	AF132984	−0.76	0.003
Nuclear pore complex interacting protein	NPIPL5	BC144439	−0.77	0.020
RNA binding motif protein 14	RBM14	BC007641	−0.66	0.001
RNA binding motif protein 14	RBM14	BC007641	−0.71	0.005
RNA binding motif protein 5	RBM5	AF091263	−0.74	0.001
SMG1 homolog, phosphatidylinositol 3-kinase-related kinase	SMG1	AB061371	−0.74	0.009
SMG1 homolog, phosphatidylinositol 3-kinase-related kinase	SMG1	AF395444	−0.76	0.006
SMG1 homolog, phosphatidylinositol 3-kinase-related kinase	SMG1	AF395444	−0.77	0.009
Coiled-coil domain containing 82	CCDC82	AK313893	−0.67	0.000
WD repeat domain 52	WDR52	AK002004	−0.70	0.022
Pyridoxal-dependent decarboxylase domain containing 2	PDXDC2	AK294177	−0.75	0.008
Coiled-coil domain containing 142	CCDC142	BC143399	−0.75	0.001
Cell cycle
Chromosome 12 open reading frame 27	C12orf27		−0.47	0.001
Chromosome 3 open reading frame 42	C3orf42	AF280797	−0.68	0.001
Chromosome 6 open reading frame 134	C6orf134	BC047303	−0.70	0.009
Chromosome 17 open reading frame 91	C17orf91	BX648321	−0.70	0.001
Chromosome 20 open reading frame 29	C20orf29	BC043344	−0.71	0.001
Chromosome 15 open reading frame 28	C15orf28		−0.73	0.002
Chromosome 2 open reading frame 49	C2orf49	AK127661	−0.73	0.004
Chromosome X open reading frame 40B	CXorf40B	L43578	−0.73	0.005
Chromosome 19 open reading frame 6	C19orf6	DQ005958	−0.74	0.000
Chromosome 6 open reading frame 64	C6orf64	BC022007	−0.75	0.004
Chromosome 2 open reading frame 14	C2orf14	AK093281	−0.77	0.039
Chromosome 17 open reading frame 88	C17orf88	AF143236	−0.76	0.016
Transcriptional regulator
Zinc finger protein 778	ZNF778	AK295122	−0.63	0.000
Zinc finger and SCAN domain containing 16	ZSCAN16	BC004255	−0.76	0.006
Zinc finger family member 767	ZNF767	BC047675	−0.76	0.013
Zinc finger, BED-type containing 3	ZBED3	BC007239	−0.77	0.005
Activating transcription factor 4	ATF4	D90209	−0.76	0.000
Activating transcription factor 5	ATF5	AB021663	−0.74	0.004
Ring finger protein 5 pseudogene 1	RNF5P1	BC119741	−0.59	0.000
Ring finger protein 5 pseudogene 1	RNF5	BC111392	−0.65	0.000
Taste transduction (Bitter taste)
Taste receptor, type 2, member 14	TAS2R14	BC103699	−0.62	0.017
Taste receptor, type 2, member 20	TAS2R20	BC100915	−0.65	0.013
Taste receptor, type 2, member 31	TAS2R31	BC117421	−0.67	0.023
Taste receptor, type 2, member 4	TAS2R4	BC130439	−0.68	0.039
Taste receptor, type 2, member 19	TAS2R19	BC101804	−0.74	0.031
Taste receptor, type 2, member 3	TAS2R3	BC095523	−0.76	0.041
Adhesion pathway
Mucin 12, cell surface associated	MUC12		−0.74	0.026
Mucin 3B, cell surface associated	MUC3B	AB038783	−0.72	0.008
Peroxisome
Solute carrier family 7, (cationic amino acid transporter, y+ system)	SLC7A11	AF252872	−0.69	0.001
Solute carrier family 7 (cationic amino acid transporter, y+ system)	SLC7A6	BC028216	−0.71	0.000
Solute carrier family 25, member 32	SLC25A32	BC021893	−0.74	0.000
Meiosis
G protein-coupled receptor 21	GPR21	BC066885	−0.68	0.002
G protein-coupled receptor 75	GPR75	AF072693	−0.74	0.016
Splicesome pathway
Splicing factor, arginine/serine-rich 1	SFRS1	BC033785	−0.77	0.001
Splicing factor, arginine/serine-rich 6	SFRS6	AK300411	−0.74	0.003
Cytosolic DNA sensing pathway
Transmembrane protein 80	TMEM80	BC008671	−0.63	0.000
Transmembrane protein 88	TMEM88	BC057812	−0.76	0.009
Cytokine production
Interleukin 17 receptor B	IL17RB	AF208111	−0.77	0.024
Neurone interaction
Gamma-aminobutyric acid (GABA) A receptor, epsilon	GABRE	BC026337	−0.65	0.011
Others
Protease, serine, 35	PRSS35	AY358661	−0.44	0.001
FRSS1829	LOC100132099	AY358798	−0.46	0.005
Oculomedin	OCLM	AF142063	−0.51	0.003
MicroRNA 221	MIR221		−0.58	0.013
Nuclear receptor coactivator 5	NCOA5	BC140836	−0.60	0.000
Ankyrin repeat domain 36B	ANKRD36B	BC125132	−0.60	0.002
Transient receptor potential cation channe	LOC100133315		−0.60	0.007
Family with sequence similarity 106, member A	FAM106A		−0.62	0.001
IGYY565	LOC100130428	BC040288	−0.62	0.005
Family with sequence similarity 106, member A	FAM106A		−0.62	0.001
F-box protein 9	FBXO9	AK095307	−0.63	0.003
Family with sequence similarity 106, member C pseudogene	FAM106C		−0.63	0.002
GALI1870	LOC100133299	AY358688	−0.66	0.004
F-box protein 17	FBXO17	AK021860	−0.75	0.008

Changed gene expressions after 20 h of treatment of L-92 cells

After the 20 h of treatment, genes belonging to the categories of cell adhesion, mitogen-activated protein (MAP) kinase, transmembrane function, immune response, metabolism, and others were mainly up-regulated (Fig. 1). Among the up-regulated genes, two dual-specificity phosphatase genes and two small Cajal body-specific RNA genes in the MAP kinase pathway, four carcinoembryonic antigen-related cell adhesion molecule genes, two amphiregulin genes, and two integrin genes for cell adhesion were up-regulated (Table 2). No significantly down-regulated genes were observed in the 20 h treatment group.

Table 2

Up-Regulated Gene Expressions in Caco-2 cells After 20 H of Treatment of Lactobacillus acidophilus L-92

Category Gene description	Gene symbol	Genbank	Fold change	P value
Up-regulated genes
Cell adhesion
Carcinoembryonic antigen-related cell adhesion molecule 7	CEACAM7	X98311	1.615	0.0082
Carcinoembryonic antigen-related cell adhesion molecule 1	CEACAM1	J03858	1.509	0.0012
Carcinoembryonic antigen-related cell adhesion molecule 5	CEACAM5	M29540	1.452	0.0093
Carcinoembryonic antigen-related cell adhesion molecule 6	CEACAM6	BC005008	1.401	0.0074
Amphiregulin	AREG	BC009799	1.567	0.0053
Amphiregulin	AREG	BC009799	1.315	0.0041
Integrin, alpha 2	ITGA2		1.310	0.0071
Integrin, alpha 6	ITGA6	BC136455	1.265	0.0119
Similar to keratin 18	LOC442249		1.359	0.0009
Mannose-binding lectin (protein C) 2, soluble (opsonic defect)	MBL2	X15422	1.279	0.0069
Leucine rich repeat containing 66	LRRC66		1.360	0.0081
Keratin 20	KRT20	BC031559	1.330	0.0043
MAP kinase pathway
Dual specificity phosphatase 6	DUSP6	BC005047	1.309	0.0029
Dual specificity phosphatase 5	DUSP5	BC062545	1.304	0.0006
Small Cajal body-specific RNA 22	SCARNA22		1.460	0.0003
Small Cajal body-specific RNA 13	SCARNA13		1.301	0.0047
Cytochrome P450, family 1, subfamily A, polypeptide 1	CYP1A1	BC023019	2.076	0.0003
G protein-coupled receptor, family C, group 5, member A	GPRC5A	AK122672	1.266	0.0055
Sprouty-related, EVH1 domain containing 1	SPRED1	BC137480	1.433	0.0009
Transmembrane protein
Matrix metallopeptidase 15 (membrane-inserted)	MMP15	BC036495	1.389	0.0029
Transmembrane channel-like 7	TMC7	BC036205	1.373	0.0089
Homeobox B9	HOXB9	BC015565	1.495	0.0004
Transmembrane protein ENSP00000340100	LOC100129969		1.337	0.0132
Immune related pathway
Fc fragment of IgE	FCER1G	M33195	1.428	0.0048
Immunoglobulin heavy constant delta	IGHD	BC021276	1.356	0.0016
Immediate early response 3	IER3	BC005080	1.274	0.0140
ERBB receptor feedback inhibitor 1	ERRFI1	BC025337	1.272	0.0026
Metabolism
Chromosome 10 open reading frame 54	C10orf54	AY358379	1.305	0.0119
Chromosome 17 open reading frame 78	C17orf78	BC034672	1.275	0.0013
Histone cluster 1, H2ai	HIST1H2AI	BC112254	1.332	0.0090
DNA binding
ets variant 4	ETV4	BC016623	1.325	0.0098
Inflammation
Acyloxyacyl hydrolase (neutrophil)	AOAH	BC025698	1.311	0.0146
Protein synthesis
Ribosomal protein S3A	RPS3A	L13802	1.652	0.0099
Others
2′,5′-oligoadenylate synthetase 1, 40/46 kDa	OAS1	AY730627	1.273	0.0002
Ankyrin repeat domain 1 (cardiac muscle)	ANKRD1	BC018667	1.428	0.0065
Tescalcin	TESC	AK000614	1.255	0.0048
Glutathione peroxidase 2 (gastrointestinal)	GPX2	BC005277	1.350	0.0024
Carbonic anhydrase XII	CA12	AF051882	1.328	0.0002
WNK lysine deficient protein kinase 4	WNK4	BC136664	1.392	0.0040
Serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1)	SERPINE1	BC010860	1.333	0.0060
ATP-binding cassette, sub-family B (MDR/TAP), member 1	ABCB1	M14758	1.466	0.0097
Transforming, acidic coiled-coil containing protein 1	TACC1	AF049910875	1.285	0.0030
Similar to hCG2042717	LOC100293211		1.565	0.0038
Recombination signal binding protein for immunoglobulin kappa J region	RBPJ	D14041	1.279	0.0108
Serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1)	SERPINE2	BC042628	1.499	0.0056
Mannosyl (beta-1,4-)-glycoprotein beta-1,4-N-acetylglucosaminyltransferase	MGAT3	BC113383	1.456	0.0023

Changed gene expressions after 4 and 20 h of treatment of L-92 cells

Only two genes in the metabolism and nucleosome structure group were significantly up-regulated after both the 4 and 20 h of treatments. Genes for meiosis, cytosolic DNA sensing, cell cycle, and leucine rich repeat were down-regulated after 4 h but up-regulated after the 20 h of treatment. Throughout the present transcriptome analysis, during the early stage of treatment with L-92 (four hours of treatment), many genes linked to cell growth and cell meiosis were mainly repressed, as shown in Figure 1. During the late phase of the treatment (20 h), many genes linked to cell adhesive activity and metabolism of cell growth, such as DNA synthesis, protein synthesis, and kinase activity, were increased (Fig. 1).

Summary of the changed gene expressions in L-92 cells

To help understand the events occurring in the Caco-2 cells after L-92 treatment, many of the genes listed in Tables 1 and 2 are illustrated in Figures 2 and 3. During the early stage of the treatment (4 h), genes such as FBX, RNF, ZNF, and ATF (Table 1) were down-regulated, and two metabolism-related genes, both involved in apoptosis, were up-regulated (Fig. 2). Moreover, genes involved in RNA splicing and RNA degradation were down-regulated, and genes encoding RNA synthesis were up-regulated (Fig. 2). Genes encoding the adhesive molecule mucin and some genes linked to transcriptional regulation and MAP kinase were down-regulated (Fig. 2). In addition, genes linked to phagocytosis and the G-protein-coupled receptor were also down-regulated. These results suggest that Caco-2 cells might respond to L-92 treatment by slowing down cell death and RNA degradation, and accelerating RNA synthesis as an early response. After the 20 h of treatment, most of the genes linked to cell proliferation, gene regulation, MAP kinase, apoptosis, and some adhesive molecules were up-regulated (Fig. 3). These results suggest that long-term treatment of L-92 (20 h) might activate cell proliferation, transcriptional activity, and apoptosis in Caco-2 cells.

FIG. 2.

Genes mainly changed in Caco-2 after 4-h exposure to L. acidophilus L-92. Down-regulated genes are indicated with blue, while up-regulated genes are red. Categorized networks are circled.

FIG. 3.

Genes mainly changed in Caco-2 cells after 20-h exposure to L. acidophilus L-92. Up-regulated genes are indicated with red. Categorized networks are circled.

Discussion

Host-bacterial cross-talk between probiotic bacteria and GI epithelial cells is thought to be an important process in eliciting the host response, including defensive responses against pathogenic bacteria and immune responses. To understand the probiotic roles of the host GI cells, transcriptome analyses for human GI cell samples collected by biopsy are indicated. However, this is not an easy task. Separations of the epithelial cells and lamina propria immune cells, which may play different roles in host defense and immunomodulatory effects, are also difficult to achieve. To mimic the in vivo gut environment, human intestinal epithelial Caco-2 cells have been utilized to study GI events, epithelial disease status, transport of nutrients, and bioactive components.^27
–29 In the present study, we performed a transcriptome analysis of epithelial Caco-2 cells to analyze the host GI response after the uptake of probiotic L-92 cells. Specific binding of the L-92 cells to Caco-2 cells was expected via a cell-wall-associated adhesive molecule, SlpA, present in L-92 cells.²⁵ Changes in gene expression profile in Caco-2 cells were relatively mild, but many significant changes were observed by microarray analysis after 4 and 20 h of treatment, as summarized in Figures 2 and 3. The results suggest that Caco-2 cells are prevented from cell death, categorized as apoptosis, and cell proliferation (growth) during the early phase of the treatment (Fig. 2). Based on these results, Caco-2 cells seem to respond first to the stress from L-92 binding, then adjust their activities to cell proliferation during the late phase of the treatment. By repeated oral uptakes of probiotic L-92 cells, responses observed in Caco-2 cells at 20 h would be most likely expected in the epithelial GI tract in humans.

Probiotic strains are thought to communicate with intestinal epithelial cells in a strain-specific manner. Some of the responses observed in this study were previously reported using different lactic acid bacterial strains in animal, human, and cultured cell studies. Among these previous studies, Lactobacillus GG was shown to suppress cytokine-induced apoptosis in in vitro cultured epithelial cells.³⁰ By contrast, up-regulation of anti-apoptotic and cytoprotective genes was confirmed in the animal study that used orally administered Lactobacillus GG.³¹ The repressive effect on apoptosis was observed in previous studies³² and in the present study during the early stage. The induction of MAP kinases, observed in this study after the 20 h of treatment, was also detected in intestinal epithelial murine colonic YAMC cells after Lactobacillus GG treatment (32). Changes in gene expression for G-protein coupled receptor, cytochrome P450, and zinc finger protein were observed in the present study and was also previously reported.¹⁷

Differential responses to probiotic bacteria between the present study and previous in vivo studies have been observed. In a previous study on the human intestinal tract, up-regulation of a G-protein coupled receptor was observed after 1 h of treatment in a clinical trial, but it was only up-regulated after 20 h of treatment in the present study.¹⁰ In a human challenge trial, various genes linked to the immunoregulatory response were also changed.¹⁰ In contrast, changes in the expression of genes involved in the immunomodulatory response were small in the present study. These results obtained in the previous in vivo studies may involve responses not only from epithelial cells but also from immune cells, such as dendritic cells and T cells. Moreover, treatment with Lactobacillus GG over a long period in the human challenge trial increased the expression of many immunomodulatory genes, such as cytokines and chemokines.⁹ However, this was not observed in the present study. In contrast, increases in the gene expression of mucin, integrin, cytochrome P450, and ubiquitin observed in this in vitro study were also detected throughout the continuous uptake of GG in the previous study.⁹ Moreover, human colon epithelial cells, such as T84 cells, HT29 cells, and Caco-2 cells, secreted pro-inflammatory and chemo-attractant cytokines in response to bacterial invasion treatment in a previous study.³³ However, the expression levels of cytokine genes were largely unchanged in the present study.

Many studies have focused on host GI responses to probiotic lactic acid bacteria, yet details of host-probiotic cross-talk are not fully understood. Probiotic strains are thought to specifically communicate with GI epithelial cells in a strain-dependent manner. Throughout the present transcriptome analysis of cultured Caco-2 cells, the host epithelial response to specific exposure with L-92 was mainly discussed to understand its impact on the GI tract. Our previous study, involving comparative proteome analysis with the L. acidophilus CP23 strain, which lacks SlpA, revealed that the binding of L-92 cells to Caco-2 cells might be strongly associated with the cell-surface SlpA protein (25). By attachment of the L-92 strain to the epithelial cells, most likely via the SlpA protein, Caco-2 cells seemed to respond to L-92 by accelerating cell proliferation after the first communication, during the early stage. It is also suggested that L-92 might have an in vivo effect on epithelial cells of the GI tract by specific binding. Information obtained in the present study could be used in considering the impact of L-92 on the GI tract and host health. In addition, comparative transcriptome analyses using different probiotic strains on Caco-2 cells would be of interest to help understand the potential of probiotics. Moreover, transcriptome analysis of immune cells, such as dendritic cells, should be addressed next in order to further understand gastric immunity.

Footnotes

Author Disclosure Statement

The authors confirm that no competing financial interests exist.

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