Archives of Oral Biology 118 (2020) 104832

Contents lists available at ScienceDirect

Archives of Oral Biology
journal homepage: www.elsevier.com/locate/archoralbio

Comprehensive analysis of transcriptional proﬁles in oral epithelial-like cells
stimulated with oral probiotic Lactobacillus spp.

T

Kimika Endoa, Yuichi Mineb,*, Takahiro Shutoc, Tsuyoshi Tajia, Takeshi Murayamab,
Hiroki NikawaaaDepartment of Oral Biology & Engineering, Division of Oral Health Sciences, Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi
Minami-ku, Hiroshima, 734-8553, Japanb
Department of Medical System Engineering, Division of Oral Health Sciences, Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi
Minami-ku, Hiroshima, 734-8553, Japanc
Department of Oral Health Engineering, Faculty of Health Sciences, Osaka Dental University, 1-4-4, Makinohonmachi, Hirakata-city, Osaka, 573-1144, JapanA R T I C LE I N FO

A B S T R A C T

Keywords:
Transcriptional proﬁling
Gingival
Epithelial cells
Lactobacillus
Probiotic

Objective: The mechanisms of action of probiotics can vary among species and among strains of a single species;
thus, they can aﬀect host cells in a complex manner. In the present study, Lactobacillus spp. were evaluated fortheir ability to adhere to gingival epithelial-like cells. Comprehensive analyses of transcriptional proﬁles ofmouse gingival epithelial GE1 cells treated with L. rhamnosus L8020 were performed to assess the putative in vivo
probiotic potential of this strain.
Methods: Five Lactobacillus spp., isolated from the oral cavity, traditional Bulgarian yoghurt, and the feces of ahealthy human, were each co-cultured with GE1 cells. Adhesion assays with serial dilution plating and DNA
microarray analysis were performed to identify diﬀerentially expressed genes (DEGs) in GE1 cells grown in coculture with L. rhamnosus L8020.
Results: The oral isolates L. rhamnosus L8020, L. casei YU3, and L. paracasei YU4 demonstrated signiﬁcantly
greater adhesion compared with the non-oral isolates. In total, 536 genes in GE1 cells exhibited more than
twofold upregulation or downregulation, compared with the 0 h timepoint, during co-culture with L. rhamnosus
L8020. Gene ontology enrichment analysis revealed that DEGs were diﬀerentially enriched in a time-dependent
manner. Early responses involved widespread changes in gene expression.
Conclusions: This study reveals changes in expression of genes involved in the epithelial physical barrier andimmune response in gingival epithelial-like cells co-cultured with L. rhamnosus L8020. Further investigations
regarding the molecular mechanisms by which L. rhamnosus L8020 serves as a probiotic may provide evidence tosupport clinical use.

1. Introduction
Probiotics are live microorganisms that can confer a health beneﬁt
to the host upon oral intake (Food & Agriculture Organization/World
Health Organization, 2001; Hill et al., 2014). Various studies have investigated the use of probiotics to prevent health problems, including
digestive disorders, allergic disorders, and dental and oral diseases(Bermudez-Brito, Plaza-Díaz, Muñoz-Quezada, Gómez-Llorente, & Gil,2012; Nikawa et al., 2011).
Microbes of the genus Lactobacillus, which ferment saccharides to
produce lactic acid, are the predominant indigenous bacteria in thehuman oral cavity, gastrointestinal tract, and vagina. Novel
Lactobacillus species are regularly described, such that the genus now
includes > 200 species (Salvetti, Harris, Felis, & O’Toole, 2018). Many
Lactobacillus species are used in fermented food production (e.g., asstarter cultures or food preservatives) and in probiotics; accordingly,
they have a long history of safe usage in foods and drug supplements(Papizadeh, Rohani, Nahrevanian, Javadi, & Pourshaﬁe, 2017). We
previously isolated 42 Lactobacillus spp. from healthy volunteers who
had no caries or caries-treatment experience. Among the 42 isolates,
ﬁve, including L. rhamnosus 8020, L. casei YU3, and L. paracasei YU4,
showed > 95 % growth inhibition of Porphyromonas gingivalis andAbbreviations: MRS, de Man Rogosa and Sharpe; CXCL, chemokine (C–X–C motif) ligand; CCL, chemokine (C–C motif) ligand; BP, biological processes; CC, cellularcomponents; MF, molecular functions; IFIT, interferon-induced protein with tetratricopeptide repeats; PAI, plasminogen activator inhibitor⁎
Corresponding author.
E-mail address: mine@hiroshima-u.ac.jp (Y. Mine).
https://doi.org/10.1016/j.archoralbio.2020.104832
Received 31 March 2020; Received in revised form 5 June 2020; Accepted 7 July 2020
0003-9969/ © 2020 Elsevier Ltd. All rights reserved.

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resuspended to a ﬁnal concentration of 1 × 108 colony-forming units/
mL. Then, 100 μL Lactobacillus spp. suspension were added to each wellof conﬂuent GE1 cells (multiplicity of infection 14.3:1).
To evaluate the adhesion of Lactobacillus spp. to GE1 cells, a serial
dilution plating method was used, with a modiﬁed version of a previously reported protocol (Wang et al., 2018). After 4 h of co-culture,
the wells were washed twice with phosphate-buﬀered saline and the
GE1 cells were lysed with 1% Triton-X (Nacalai Tesque, Kyoto, Japan).
Numbers of viable adherent Lactobacillus spp. were determined by serial
plating on MRS-agar (Difco).

Streptococcus sobrinus in vitro. L. rhamnosus 8020 also showed > 95 %growth inhibition of Candida albicans. On the basis of this in vitro study,
we performed clinical studies and found that L. rhamnosus 8020 could
reduce the oral burden of mutans streptococci and the risk of periodontal disease (Nikawa et al., 2011; Yuki et al., 2019), suggesting thatdaily consumption of these probiotic organisms may be useful for prevention of dental and oral diseases.
Pathogenic bacteria are well known to induce altered gene expression in host cells (Handﬁeld et al., 2005; Zhu, Lu, Wei, Cai, & Wang,2018). Notably, multiple studies have shown that probiotics are alsoable to modulate host gene expression (de Andrés, Jiménez, EspinosaMartos, Rodríguez, & García-Conesa, 2018; Plaza-Díaz et al., 2017;
Taranu et al., 2018). In the oral cavity, epithelial cells act as a physicalbarrier against invasive and commensal microorganisms. Gingival epithelial cells may be aﬀected by invasive pathogenic microorganisms(e.g., P. gingivalis, Aggregatibacter actinomycetemcomitans, and C. albicans) and their protective function may be modulated by probiotics.
The mechanisms of action of probiotics can vary among species andamong strains of a single species; thus, they can aﬀect host cells in acomplex manner (Tsilingiri et al., 2012). Accordingly, research regarding probiotic mechanisms in the oral cavity can facilitate selectionof strains for clinical and/or commercial use in treatment or preventionof particular pathological conditions. In the ﬁrst part of the present
work, three oral isolates of Lactobacillus spp. were evaluated for theirability to adhere to gingival epithelial-like cells; these oral isolates
showed signiﬁcantly greater adhesion compared with non-oral isolates.
In the latter part of the study, one oral isolate, L. rhamnosus L8020,
which has the widest antimicrobial spectrum among the three oral
isolates used in this study, was analyzed to determine its ability to alter
the transcriptional proﬁle of gingival epithelial-like cells and contributeto host defense, indicating its putative probiotic potential.

2.4. DNA microarray analysis
Expression proﬁles were determined using the Mouse Clariom™ S
Assay (Thermo Fisher Scientiﬁc, Waltham, MA, USA). GE1 cells weregrown to conﬂuence on 24-well plates, then co-cultured with
Lactobacillus spp. for 1 or 4 h. Total RNA was isolated from GE1 cells
using an RNeasy Mini Kit (Qiagen, Hilden, Germany). cDNA was synthesized using a GeneChip WT Ampliﬁcation kit (Thermo Fisher
Scientiﬁc) following the manufacturer’s instructions. The sense cDNA
was then fragmented and biotin-labeled with terminal deoxynucleotidyltransferase using a GeneChip WT Terminal labeling kit (Thermo Fisher
Scientiﬁc). Approximately 5.5 μg of labeled DNA target was hybridized
to the Aﬀymetrix GeneChip Array at 45 °C for 16 h. Hybridized arrays
were washed and stained on a GeneChip Fluidics Station 450 andscanned on a GCS3000 Scanner (Thermo Fisher Scientiﬁc). The data
were summarized and normalized using the Signal SpaceTransformation-Robust Multichip Analysis method implemented inAﬀymetrix Power Tools (Thermo Fisher Scientiﬁc). Statistical signiﬁcance of expression data was determined by using fold-change
analysis. To identify diﬀerentially expressed genes, hierarchical cluster
analysis was performed with MultiExperiment Viewer, version 4.9.0,
using complete linkage and Euclidean distance as a measure of similarity. Gene enrichment and functional annotation analysis to identify
signiﬁcant terms was performed using Gene Ontology (http://
geneontology.org); p < 0.005 was regarded as the threshold value.

2. Materials and methods
2.1. Bacterial strains
L. rhamnosus L8020, L. casei YU3, and L. paracasei YU4, previously
isolated from healthy and caries-free volunteers (clinical isolates,
Hiroshima University Hospital, Hiroshima, Japan) (Nikawa et al., 2011)were selected as the probiotics for investigation in this study. L. gasseri
OLL2716 and L. delbrueckii ssp. bulgaricus OLL1073R-1 were used as
non-oral isolates. L. gasseri OLL2716 was isolated from the feces of ahealthy human (Kimura, 2011); L. delbrueckii ssp. bulgaricus OLL1073R1 was originally isolated from traditional Bulgarian yoghurt (Makino
et al., 2010). Lactobacillus spp. were preincubated in Lactobacillus deMan, Rogosa and Sharpe (MRS; Difco, Tokyo, Japan) broth for 24 h at
37 °C.

2.5. Statistical analysis
One-way analysis of variance and Tukey’s multiple range test wereused to analyze diﬀerences among groups. Statistical analyses were
performed using SPSS Statistics software, version 21.0 (IBM Corp.,
Armonk, NY, USA).
3. Results
3.1. Oral-isolate Lactobacillus spp. adhere to GE1 cells
As shown in Fig. 1, serial dilution plating revealed that L. rhamnosus
L8020, L. casei YU3, and L. paracasei YU4 demonstrated signiﬁcantly
greater adhesion to mouse GE1 cells than non-oral isolates L. gasseri
OLL2716 and L. delbrueckii ssp. bulgaricus OLL1073R-1 (p < 0.05,
p < 0.01).

2.2. Cell culture
The GE1 mouse gingival epithelial cell line was obtained fromRIKEN Bioresource Center Cell Bank (RCB1709; Ibaraki, Japan)(Hatakeyama et al., 2001; Mine et al., 2010). GE1 cells were previously
shown to display a gingival epithelial cell phenotype (Makihira et al.,2010). Cells were cultured in antibiotic-free SFM-101 (Nissui, Tokyo,
Japan) containing 1 % fetal bovine serum (HyClone, South Logan, UT,USA) and 10 ng/mL mouse epidermal growth factor (Sigma-Aldrich, St.
Louis, MO, USA); cells were incubated at 33 °C in 5% CO2/95 % air.

3.2. Identiﬁcation of diﬀerentially expressed genes in GE1 cells co-cultured
with L. rhamnosus L8020
To investigate the gene expression proﬁles underlying putative
probiotic eﬀects, DNA microarray analysis was performed in GE1 cells
after 0, 1, and 4 h of co-culture with L. rhamnosus L8020.
Transcriptional proﬁles observed after 1 h of co-culture were deﬁned as
early responses; proﬁles observed after 4 h of co-culture were deﬁned as
secondary responses. In total, 536 genes exhibited more than twofold
upregulation or downregulation compared with the 0-h timepoint
during co-culture (Fig. 2A); deﬁned early responses included 476 genes
with changes in expression. Deﬁned secondary responses included 103

2.3. Adhesion of Lactobacillus spp. to GE1 cells
GE1 cells were seeded onto 24-well plates at 2 × 104 cells/well, andthe culture medium was changed on day 3. After reaching conﬂuency,
there were 7 × 105 GE1 cells per well. Lactobacillus spp. cells were
harvested during the exponential growth phase by centrifugation at
1000×g, washed twice with phosphate-buﬀered saline (pH 6.8), and2

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Fig. 1. Adhesion to GE1 cells, compared between Lactobacillus rhamnosus L8020(L8020), L. casei YU3 (YU3), L. paracasei YU4 (YU4), L. gasseri OLL2716(OLL2716), and L. delbrueckii ssp. bulgaricus OLL1073R-1 (OLL1073R-1). GE1
cells were co-cultured with Lactobacillus spp. for 4 h. Data in all graphs represent the means ± standard deviations of three independent cultures.
Asterisks indicate statistically signiﬁcant diﬀerences between Lactobacillus spp.
(*p < 0.05, **p < 0.01).

genes with changes in expression. Forty-three genes overlapped between the 476 (early responder) and 103 (secondary responder) genes(Fig. 2A, B).
Early responses included upregulation of genes such as encoding
Claudin-1, Desmocollin-2, and Desmocollin-3, all of which are involvedin epithelial barrier function (Table 1). Downregulated genes identiﬁed
in early responses are shown in Table 2. Upregulated genes identiﬁed insecondary responses included chemokine (C–X–C motif) ligand (CXCL)
1, CXCL10, and chemokine (C–C motif) ligand (CCL) 2; all of these are
known immunomodulatory chemokines (Table 3). Downregulated
genes identiﬁed in secondary responses are shown in Table 4.

Fig. 2. Comprehensive DNA microarray analysis of mouse GE1 cells co-cultured
with L. rhamnosus L8020. To identify L. rhamnosus L8020-responsive genes, GE1
cells were co-cultured with L. rhamnosus L8020 for 1 or 4 h. (A) Venn diagram
illustrating subsets of altered genes among 536 genes with signiﬁcant change in
expression (fold change > 2) compared with the 0-h timepoint. (B) Heatmap of
microarray data.

3.3. Gene ontology enrichment analysis of GE1 cells co-cultured with L.
rhamnosus L8020

Table 1
Top 20 upregulated genes at 1 h.

Gene ontology analysis identiﬁed 712 terms that were signiﬁcantly
enriched at the 1 h co-culture timepoint, including biological processes(BP), cellular components (CC), and molecular functions (MF). The top
10 Gene Ontology terms in each sub-ontology (i.e. BP, CC, and MF) are
shown in Fig. 3A. Moreover, 383 terms were signiﬁcantly enriched atthe 4 h co-culture timepoint; the top 10 Gene Ontology terms in each
sub-ontology are shown in Fig. 3B. Co-culture with L. rhamnosus L8020
for 1 h had potential impacts on numerous BP, including system development, anatomical structure morphogenesis, anatomical structuredevelopment, animal organ development, and multicellular organism
development. Furthermore, co-culture with L. rhamnosus L8020 for 4 h
had potential impacts on response to cytokines, defense response, response to biotic stimulus, response to another organism, and response
to external biotic stimulus.

Gene symbol

Description

Fold change

Ntrk2
Cldn1
Txnip
Arrdc3
Fam83b
Kitl
Dusp6
Spp1
Dbp
Spry1
Gm17651neurotrophic tyrosine kinase, receptor, type 2
claudin 1thioredoxin interacting protein
arrestin domain containing 3
family with sequence similarity 83, member B
kit ligand
dual speciﬁcity phosphatase 6
secreted phosphoprotein 1
D site albumin promoter binding protein
sprouty homolog 1 (Drosophila)
predicted gene, 17651 [Source:MGI
Symbol;Acc:MGI:4937285]
desmocollin 2
muscular LMNA-interacting protein
frizzled homolog 4 (Drosophila)
desmocollin 3
early growth response 1
dedicator of cytokinesis 8
ets variant 5
basic helix-loop-helix family, member e41
trichorhinophalangeal syndrome I (human)

13.698443
7.434419
7.419490
6.339988
5.561022
5.463896
4.746256
4.729278
4.064140
3.813564
3.645104

Dsc2
Mlip
Fzd4
Dsc3
Egr1
Dock8
Etv5
Bhlhe41
Trps1

4. Discussion
Microbes in the oral cavity coexist with the host in a mostly harmonious symbiotic relationship. However, bioﬁlm accumulation can
act as a triggering event, which results in complex immune interactions
that induce gingivitis and lead to periodontitis (Kilian et al., 2016). The
oral epithelial barrier is composed of two main systems: a physicalbarrier with an intact epithelial multilayer and intracellular junctions,33.614184
3.589219
3.487033
3.485752
3.435000
3.284947
3.215092
3.211706
3.163628

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Table 2
Top 20 downregulated genes at 1 h.

Table 4
Top 20 downregulated genes at 4 h.

Gene symbol

Description

Fold change

Gene symbol

Description

Fold change

Ctgf
Il1rn
Abi3bpconnective tissue growth factor
interleukin 1 receptor antagonist
ABI gene family, member 3 (NESH) binding
proteinpiezo-type mechanosensitive ion channel
component 2
T cell speciﬁc GTPase 2
prostate transmembrane protein, androgen
induced 1
stratiﬁn
predicted gene 12185
tensin 1
serine (or cysteine) peptidase inhibitor, clade E,
member 1
transgelin
WNT1 inducible signaling pathway protein 1
cytochrome P450, family 1, subfamily b,
polypeptide 1
chemokine (C-C motif) ligand 5
family with sequence similarity 101, member A
lysyl oxidase-like 2
predicted gene 11096 [Source:MGI
Symbol;Acc:MGI:3779332]
inosine 5-phosphate dehydrogenase 1
ﬁbulin 5
interferon-induced protein with tetratricopeptide
repeats 1−14.995939−11.316924−10.077808

Gjb3
Ctgf
Rnf17
Serpine1

gap junction protein, beta 3
connective tissue growth factor
ring ﬁnger protein 17
serine (or cysteine) peptidase inhibitor, clade E,
member 1
myelin regulatory factor-like
olfactory receptor 23
tensin 1
ubiquitin-conjugating enzyme E2D 2B
olfactory receptor 978
MOK protein kinasepredicted gene 11084 [Source:MGI
Symbol;Acc:MGI:3779315]
oxidative stress induced growth inhibitor 1
grainyhead-like 3 (Drosophila)
runt related transcription factor 1
RIKEN cDNA 6030408B16 genefamily with sequence similarity 19, member A1
prostate transmembrane protein, androgen
induced 1
heparan sulfate 6-O-sulfotransferase 3
glycoprotein hormones, alpha subunit
predicted gene, 17482 [Source:MGI
Symbol;Acc:MGI:4937116]−2.497042−2.366672−2.326998−2.315173

Piezo2
Tgtp2
Pmepa1
Sfn
Gm12185
Tns1
Serpine1
Tagln
Wisp1
Cyp1b1
Ccl5
Fam101a
Loxl2
Gm11096Impdh1
Fbln5
Iﬁt1−8.759448
Myrﬂ
Olfr23Tns1
Ube2d2b
Olfr978
Mok
Gm11084−7.712775−7.656049−7.640780−6.480746−6.113167−5.926786

Osgin1Grhl3
Runx1
6030408B16Rik
Fam19a1
Pmepa1−5.811498−5.743141−5.595744−5.506135−5.372822−5.352897−5.293053

Hs6st3Cga
Gm17482−5.244263−5.176439−5.066291

Description

Fold change

Cxcl1
Cxcl5
Iﬁt1

chemokine (C-X-C motif) ligand 1
chemokine (C-X-C motif) ligand 5interferon-induced protein with tetratricopeptide
repeats 1
chemokine (C-C motif) ligand 2
nuclear factor of kappa light polypeptide geneenhancer in B cells inhibitor, zeta
chemokine (C-X-C motif) ligand 10
zinc ﬁnger CCCH type containing 12A
guanylate binding protein 2b
chemokine (C-C motif) ligand 7
interferon-induced protein with tetratricopeptide
repeats 3B
interferon-induced protein with tetratricopeptide
repeats 3
immunity-related GTPase family M member 2
ubiquitin speciﬁc peptidase 18
2-5 oligoadenylate synthetase-like 2tumor necrosis factor, alpha-induced protein 3
nitric oxide synthase 2, inducible
vascular cell adhesion molecule 1
nuclear factor of kappa light polypeptide geneenhancer in B cells inhibitor, epsilon
nuclear factor of kappa light polypeptide geneenhancer in B cells inhibitor, alphapredicted gene 4951

19.687527
12.676196
9.113214

Ccl2
NfkbizCxcl10Zc3h12a
Gbp2b
Ccl7
Iﬁt3b
Iﬁt3
Irgm2
Usp18
Oasl2
Tnfaip3
Nos2
Vcam1
NfkbieNfkbiaGm4951−2.155944−2.114007−2.091677−2.083083−2.074293−2.048517−2.041472−2.034945−2.017740

Moreover, a clinical study implied that L. rhamnosus SD11 (derived
from the human oral cavity) may exhibit better adhesion to the gingival
epithelium than non-oral strains of human origin (Rungsri et al., 2017).
Consistent with these prior results, we conﬁrmed that L. rhamnosus
L8020, L. casei YU3, and L. paracasei YU4 exhibited better adhesion to
GE1 cells than did non-oral strains of human origin (i.e., L. gasseri
OLL2716 and L. delbrueckii ssp. bulgaricus OLL1073R-1).
The mechanisms underlying the ability of L. rhamnosus L8020 toreduce the oral burden of mutans streptococci and the risk of periodontal disease have not been fully elucidated. Our comprehensive transcriptomic analysis revealed that deﬁned early responses to L. rhamnosus L8020 involved widespread changes in gene expression, which
supported reinforcement of the physical barrier in healthy gingival
epithelial-like cells. Claudins, a multigene family with at least 27
members in humans and mice, are the main components of tightjunctions. Claudin-1, a major component of tight junctions in the oral
epithelium (Belibasakis, Kast, Thurnheer, Akdis, & Bostanci, 2015;
Fujita et al., 2012), was signiﬁcantly upregulated as an early response
as deﬁned in the present study. Notably, Claudin-1 has been identiﬁed
as a crucial component for physical barrier function; experimental
periodontal disease causes reduced expression of Claudin-1, suggesting
involvement in the initiation of periodontal disease (Fujita et al., 2012).
Desmocollins constitute desmosomes in combination with desmogleins;notably, desmocollins (Desmocollin-2 and Desmocollin-3) were signiﬁcantly upregulated as early responses. Desmocollins play crucial
roles in cell–cell junctions; furthermore, DNA microarray and/or immunostaining analysis demonstrated low desmocollin expression levels
in human periodontitis tissues compared with healthy tissues (Gürsoy
et al., 2016; Kim, Ramoni, Nevins, & Fiorellini, 2006).
Regarding deﬁned secondary responses, co-culture with L. rhamnosus L8020 treatment served to reinforce the immune barrier in thepresent study. The immunomodulatory properties of probiotics havebeen clearly elucidated in gut tissue through in vitro, in vivo, and clinicalstudies (van Baarlen et al., 2011). Some previous investigations showedthat exposure to probiotic Lactobacillus spp. led to upregulation ofchemokines (e.g., CXCL1, CXCL10, CCL2, and interleukin-8) in humanintestinal epithelial cells and in vivo healthy adult mucosa (O’Callaghan,
Buttó, MacSharry, Nally, & O’Toole, 2012; O’Flaherty & Klaenhammer,
2012; van Baarlen et al., 2009, 2011). Despite tissue-related and cellrelated diﬀerences, the results from our in vitro model and other

Table 3
Top 20 upregulated genes at 4 h.
Gene symbol−2.306300−2.299818−2.289623−2.225979−2.219293−2.213716−2.175188

8.344930
7.305181
6.735243
6.432190
6.385059
6.291439
5.855003
5.395101
4.609371
4.560810
4.5451254.3071094.224147
3.998974
3.947447
3.852540
3.605052

and an immune barrier that maintains immune responses or immune
tolerance to microbial antigens (Wang et al., 2019). Thus, a clear understanding of interactions between the gingival epithelium and probiotics is important for the maintenance of oral health.
The ability to adhere to the gingival epithelium is a critical probiotic
function, which varies among bacteria and host tissues. Notably, theprobiotic L. rhamnosus GG (ATCC 53103; isolated from human feces)and most yoghurt starter lactobacilli cannot adhere to saliva-coatedhydroxyapatite and microtiter plates, which are used to simulate theoral environment (Stamatova, Kari, Vladimirov, & Meurman, 2009).

4

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Fig. 3. Gene ontology analysis revealed enriched terms based on diﬀerentially expressed genes in GE1 cells co-cultured with L. rhamnosus L8020 for (A) 1 h or (B) 4 h.
The top 10 Gene Ontology terms in each sub-ontology are shown.

5. Conclusions

investigations suggest that probiotics can regulate the expression of
immunomodulatory genes; moreover, the induction of transient epithelial cell activation may aid in maintenance of mucosal homeostasis.
The interferon-induced protein with tetratricopeptide repeats (IFIT)
family includes antiviral proteins that can inhibit viral replication bymeans of an interferon-dependent innate immune response. Notably,
expression levels of IFIT1, IFIT3, and IFIT3b were signiﬁcantly upregulated as secondary responses as deﬁned in the present study. Although the cell types are diﬀerent from gingival epithelial cells, Lactobacillus spp. can induce IFIT-1 activation in mouse cells and humanmacrophages (Gutierrez-Merino, Isla, Combes, Martinez-Estrada, &Maluquer De Motes, 2020). Some studies have shown interactions between external stimuli and gingival epithelial cells that result in
downregulation of IFIT family expression levels. Co-culture of a humanbuccal epithelial carcinoma cell line with C. albicans led to downregulation of IFIT1, IFIT3, and IFIT5 (Moyes et al., 2014). Moreover,
exposure to cigarette smoke led to downregulation of the expression
levels of IFIT1, IFIT2, IFIT3, and IFIT5 in human oral keratinocytes(Rajagopalan et al., 2018).
CTGF/CCN2 was a noteworthy gene that was downregulated in bothdeﬁned early and secondary responses in the present study; this geneencodes a secretory protein, which was originally isolated from conditioned medium of human vascular endothelial cells. In previous studies, the expression level of CTGF/CCN2 was upregulated in gingival
cells of patients with periodontitis, in a manner similar to the expression of TGF-β (Mize, Sundararaj, Leite, & Huang, 2015). Plasminogenactivator inhibitor (PAI)-1, encoded by Serpine1, was also downregulated at both the 1- and 4-h timepoints. Multiple studies have revealed elevated PAI-1 protein levels in plasma of patients with periodontitis, in a manner associated with disease progression (Akman,
Fentoğlu, Yılmaz, & Arpak, 2012; Bizzarro et al., 2007). However,analysis of gingival tissues obtained from periodontal surgery revealed
that expression levels of PAI-1 were comparable between healthy andinﬂamed epithelial tissues (Xiao, Bunn, & Bartold, 1998).
In vivo rodent models have been used to clarify novel mechanisms
and interactions between probiotic bacteria and oral diseases such asoral candidiasis (Ishijima et al., 2012), periodontal disease (Maekawa &Hajishengallis, 2014), and caries (Hillman, McDonell, Cramm, Hillman,& Zahradnik, 2009). We believe that applying the transcriptional proﬁles generated in this study to rodent models and further analysis will
be useful for clarifying the molecular mechanisms of L. rhamnosus
L8020 as a probiotic.

The probiotic potential of L. rhamnosus L8020 was investigated bymeans of transcriptional proﬁling analyses in mouse gingival epitheliallike GE1 cells. Early and secondary responses in the present study revealed changes in expression of genes involved in the epithelial physicalbarrier and immune response. Notably, early responses involved
widespread changes in gene expression. Moreover, L. rhamnosus L8020
was able to regulate the expression of immunomodulatory genes; theinduction of transient epithelial cell activation may also aid in maintenance of mucosal homeostasis. Several of the aﬀected genes may bepromising targets for maintenance and promotion of oral health using
probiotics. The present study does not elucidate the molecular mechanism of L. rhamnosus L8020 eﬃcacy in humans. However, the results provide important suggestions for further studies to clarify thedetailed mechanism. It should be emphasized that we have deliberately
used gingival epithelial-like mouse cells rather than human cell lines toapply the generated transcriptional proﬁles to rodent models. In
keeping with the present study, we will perform comparative in vitro
and in vivo experiments to elucidate the molecular mechanisms of action by which L. rhamnosus L8020 serves as a probiotic, which may
provide evidence to support clinical use.

CRediT authorship contribution statement
Kimika Endo: Conceptualization, Data curation, Formal analysis,
Investigation, Methodology, Software, Validation, Visualization,
Writing - original draft, Writing - review & editing. Yuichi Mine:
Conceptualization, Formal analysis, Investigation, Methodology,
Project administration, Resources, Software, Validation, Visualization,
Writing - original draft, Writing - review & editing. Takahiro Shuto:
Validation, Writing - review & editing. Tsuyoshi Taji: Investigation,
Validation, Writing - review & editing. Takeshi Murayama:
Supervision, Writing - review & editing. Hiroki Nikawa:
Conceptualization, Methodology, Project administration, Resources,
Supervision, Writing - review & editing.

Declaration of Competing Interest
H.N. has patents for L. rhamnosus L8020, L. casei YU3, and L.
paracasei YU4. All other authors declare no conﬂicts of interest.

5Archives of Oral Biology 118 (2020) 104832

K. Endo, et al.

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This study was partially supported by grants-in-aid from the
Ministry of Education, Culture, Sports, Science and Technology ofJapan to Y.M. and H.N. [16H05535] and Y. M. [17K17181].
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