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Characterization of the immunomodulatory andanti-Helicobacter pylori properties of the humangastric isolate Lactobacillus rhamnosus UCO-25A

Valeria Garcia-Castillo, Ana María Marín-Vega, Alejandra Ilabaca, LeonardoAlbarracín, Guillermo Marcial, Haruki Kitazawa, Apolinaria Garcia-Cancino &Julio Villena

To cite this article: Valeria Garcia-Castillo, Ana María Marín-Vega, Alejandra Ilabaca, LeonardoAlbarracín, Guillermo Marcial, Haruki Kitazawa, Apolinaria Garcia-Cancino & Julio Villena (2019):Characterization of the immunomodulatory and anti-Helicobacter�pylori properties of the humangastric isolate Lactobacillus�rhamnosus UCO-25A, Biofouling

To link to this article: https://doi.org/10.1080/08927014.2019.1675153

Published online: 24 Oct 2019.

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Characterization of the immunomodulatory and anti-Helicobacter pyloriproperties of the human gastric isolate Lactobacillus rhamnosus UCO-25A

Valeria Garcia-Castilloa,b,c, Ana Mar�ıa Mar�ın-Vegac, Alejandra Ilabacaa, Leonardo Albarrac�ınb,c,d, GuillermoMarciale, Haruki Kitazawac,f, Apolinaria Garcia-Cancinoc and Julio Villenab,c

aLaboratory of Bacterial Pathogenicity, Faculty of Biological Sciences, University of Concepcion, Concepcion, Chile; bLaboratory ofImmunobiotechnology, Reference Centre for Lactobacilli (CERELA-CONICET), Tucuman, Argentina; cFood and Feed ImmunologyGroup, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan; dLaboratory of Computing Science, Faculty ofExact Sciences and Technology, Tucuman University, Tucuman, Argentina; eLaboratory of Technology, Reference Centre forLactobacilli (CERELA-CONICET), Tucuman, Argentina; fInternational Education and Research Center for Food and AgriculturalImmunology (CFAI), Graduate School of Agricultural Science, Tohoku University, Sendai, Japan

ABSTRACTThe ability to form biofilms and the potential immunomodulatory properties of the humangastric isolate Lactobacillus rhamnosus UCO-25A were characterized in vitro. It was demon-strated that L. rhamnosus UCO-25A is able to form biofilms on abiotic and cell surfaces, andto modulate the inflammatory response triggered by Helicobacter pylori infection in gastricepithelial cells and THP-1 macrophages. L. rhamnosus UCO-25A exhibited a substantial anti-inflammatory effect in both cell lines and improved IL-10 levels produced by challengedmacrophages. Additionally, UCO-25A protected AGS cells against H. pylori infection with ahigher pathogen inhibition when a biofilm was formed. Given the importance of inflamma-tion in H. pylori-mediated diseases, the differential modulation of the inflammatory responsein the gastric mucosa by an autochthonous strain is an attractive alternative for improvingH. pylori eradication and reducing the severity of the diseases that arise from the resultingchronic inflammation.

ARTICLE HISTORYReceived 2 May 2019Accepted 27 September 2019

KEYWORDSGastric epithelial cells;gastric inflammation;Helicobacter pylori;macrophages; LactobacillusrhamnosusUCO-25A; biofilm

Introduction

Helicobacter pylori infects more than half of thehuman population worldwide and has been associatedwith serious human diseases (Bauer and Meyer 2011;Backert et al. 2016). Within the broad repertoire ofclinical presentations in H. pylori infection, is notpossible yet to predict the outcome of the disease(Cizginer et al. 2014). While most infected personsremain asymptomatic, around 10-20% develop pepticulcers and 1% gastric cancer (Shah 2015; Formanet al. 2017). H. pylori triggers a strong inflammatoryresponse that is considered a determinant factor inthe pathophysiological events associated to the infec-tion by this pathogen (Naito and Yoshikawa 2002;Rolig et al. 2013; White et al. 2015). Virulence factorsfrom H. pylori with the ability to trigger stronginflammatory responses including CagA and VacAhave been related with gastric pathogenesis (Kauret al. 2010; Da Costa et al. 2015). Moreover, the col-onization of specific H. pylori strains has been relatedwith host genetic polymorphisms which in turn are

related to high level of pro-inflammatory cytokinesrelease, gastric mucosa inflammation and the develop-ment of subsequent premalignant lesions (Rad et al.2004; Atherton 2006).

Interestingly, the participation of microbial com-munities in shaping gastric inflammation has beenoutlined recently (Brawner et al. 2017). Microbiotadysbiosis has a strong connection with different typesof inflammatory diseases affecting mucosal (intestineand lung) and non-mucosal tissues (Garc�ıa-Castilloet al. 2016; Chen et al. 2017). Similarly, research sug-gested that variations in gastric microbial populationscould have an important effect on H. pylori infection,mainly affecting the immune responses in the gastricmucosa (Rolig et al. 2013). Research found that varia-tions in mice gastrointestinal microbiota could deter-mine the gastric inflammatory outcome. Comparativestudies performed in mice in which pathogen strain,infecting dose, host genetics, husbandry, gender, andage were controlled variables, clearly demonstrateddifferences in H. pylori related inflammatory

CONTACT Apolinaria Garcia-Cancino apgarcia@udec.cl Julio Villena jcvillena@cerela.org.ar� 2019 Informa UK Limited, trading as Taylor & Francis Group

BIOFOULINGhttps://doi.org/10.1080/08927014.2019.1675153

responses (Rolig et al. 2013). In line with those find-ings, a study performed in transgenic insulin-gastrinmice which develop spontaneous atrophic gastritisand gastrointestinal intraepithelial neoplasia, showedthat alterations of microbiota accelerated H. pyloriinduced inflammation and neoplasia (Lofgren et al.2011). On the other hand, studies in H. pylori infectedand non-infected children described that a tolerogenicgastric mucosa is characteristic of young hostsinfected with the gastric pathogen. H. pylori-infectedchildren showed higher expression of Foxp3, IL-10,and TGF-b in the gastric mucosa and these immuno-logical changes were directly related to microbiotavariations (Brawner et al. 2017). These observationsare of importance because they allow the hypothesisthat the appropriate manipulation of the gastricmicrobiota could be an interesting alternative toreduce the severity of H. pylori-mediated inflamma-tory diseases.

The most abundant commensal gastric bacteria inchildren and adults belong to the Actinobacteria,Bacteroidetes, Firmicutes, Fusobacteria andProteobacteria. Some studies have found the presenceof Lactobacillus spp. in the gastric mucosa (Roos et al.2005; Garcia et al. 2009; Delgado et al. 2015) and,therefore these bacteria have been proposed as poten-tial probiotic candidates to prevent or treat H. pyloriinfection (Patel et al. 2014; Nair et al. 2016). Researchhas demonstrated that various species of Lactobacillusare capable of improving the resistance against H.pylori. However, most of these studies have been per-formed with commercially available strains of differ-ent origins (Sakamoto et al. 2001; Pe~na andVersalovic 2003; Sgouras et al. 2004; Sunanliganonet al. 2012), but few studies have evaluatedLactobacillus strains isolated from human stomach.Previously, a negative Pearson correlation betweenLactobacillus spp. and H. pylori colonization in gastricbiopsies was reported by the authors’ group, indicat-ing that the co-existence of both species is low in thegastric mucosa of symptomatic patients (Garc�ıa et al.2012). Moreover, the potential probiotic properties ofthe human gastric isolate Lactobacillus rhamnosusUCO-25A were characterized in vitro including itsresistance to pH and bile salts (Garcia et al. 2009).However, no deeper studies were performed with theUCO-25A strain in the context of H. pylori infection.Therefore, the aim of this work was to evaluate thebiofilm formation ability of L. rhamnosus UCO-25Aas well as its capacity to differentially modulate theinflammatory response triggered by H. pylori infection

in both non-immune (gastric epithelial cells) andimmune (macrophages) cells in vitro.

Materials and methods

Microorganisms

Bacterial strains were obtained from the BacterialPathogenicity Laboratory culture collection atUniversity of Concepci�on (Concepci�on, Chile). L.rhamnosus UCO-25A was isolated from humanhealthy gastric tissue and characterized in previousworks (Garcia et al. 2009). L. rhamnosus GG waskindly provided by Dr Martin Gotteland from theUniversity of Chile (Santiago, Chile). L. rhamnosusUCO-25A and L. rhamnosus GG were cultured inMann-Rogosa Sharpe broth (MRS Difco) at 37 �C for16 h, followed by culturing on MRS agar under sameconditions. Isolated colonies were suspended in phos-phate buffered saline (PBS) and adjusted to concen-trations to test in cell cultures (105, 107, 108, 109

cells ml�1).Commercially available H. pylori ATCC43504

(American Type Culture Collection, Manassas, VA,USA) was used. The H. pylori strain possesses cagPAI (CagA positive) and vacAs1a/m1. Bacterial cellswere cultured in Columbia blood agar base (Oxoid,Basingstoke, UK) supplemented with 5% horse blood(GE Healthcare) and selective supplement DENT(Oxoid) in a microaerobic atmosphere (10% CO2, 5%O2, 85% N2) at 37 �C for 72 h. Bacterial colonies weresuspended in PBS enriched with 5% horse serum (GEHealthcare). A final concentration of 6� 107 cellsml�1 was used for cell infection.

Cell lines

AGS (human gastric adenocarcinoma epithelial cells)were provided by the Bacterial PathogenicityLaboratory, Department of Microbiology, Universityof Concepcion (Chile), and stored at �80 �C.Propagation was carried out according to the thawing,propagation and freezing ATCC CRL-1739 protocolfor AGS cells. Briefly, cells were cultured in DMEM(Dulbecco’s modified Eagles Medium-Gibco) mediumsupplemented with 10% v/v of inactivated fetal bovineserum (FBS) (Biological Industries), 100U ml�1 ofpenicillin, and 100lg ml�1 of streptomycin (Corning)in T75 flasks (SPL Life Sciences). Cells were culturedat 37 �C in a 5% CO2 humidified atmosphere up to80–90% confluence. Cells (7� 104 cells ml�1) wereincubated for 24 h and media replaced with antibioticfree medium for co-culture assays.

2 V. GARCIA-CASTILLO ET AL.

The THP-1 (human monocytic leukemia) cell linewas provided by the Department of ClinicalBiochemistry and Immunology, University ofConcepci�on (Concepci�on, Chile). The thawing, propa-gation and freezing ATCC TIB-202 protocol for THP-1 cells were used. Briefly, cells were maintained inRPMI-1640 (Roswell Park Memorial Institute)medium (Gibco) supplemented with 10% v/v of inac-tivated fetal bovine serum (FBS) (BiologicalIndustries), 100U ml�1 of penicillin, and 100lg ml�1

of streptomycin (Corning). The medium was replen-ished every 3 days in order to maintain the cell con-centration at 1� 106 cells ml�1. Then, 1� 105 THP-1cells ml�1 were seeded in 24-well plates and incu-bated with 100 nM of PMA (Phorbol 12-Myristate 13-Acetate) (Sigma) for 24 h, to induce the differentiationof monocytes into macrophages. After differentiation,cells were washed to remove non-adherent cells andfresh medium without antibiotics was added. A lipo-polysaccharide (LPS) challenge was used to induceinflammation in differentiated THP1-cells. LPS wasobtained from Escherichia coli O55:B5 and preparedby phenol extraction followed by gel-filtration chro-matography (Sigma, St Louis, MO, USA LPS).

Biofilm formation assay on abiotic surfaces

In order to evaluate the biofilm forming ability underdifferent conditions, bacterial suspensions were pre-pared in bacterial broths MRS or BHI (Brain HeartInfusion BD-Bacto) supplemented with 2% v/v of glu-cose or, in the cell culture media DMEM supple-mented with 10% FBS. Bacterial suspensions(1.5� 108 or 1.5� 109 cells ml�1) were seeded in 24well-flat bottom plates, including a sterile circularglass coverslip (Fisher Scientific) in the bottom. Plateswere incubated at 10% CO2, 5% O2 y 85% N2 atmos-phere for 0, 4, 8, 12, 24 or 48 h. After each incubationtime, medium and non-attached bacteria were care-fully removed.

Biofilm formation was evaluated by the crystal vio-let staining method with modifications as previouslydescribed (Salas-Jara et al. 2016). Briefly, plates werewashed three times with 1� phosphate buffer saline(PBS), allowed to dry at room temperature for 10minand 800ll of 0.1% w/v crystal violet (Merck) wereadded for 5min. After staining, unbound dye was dis-carded and wells were washed four times with PBS.Finally, biofilm attached dye was eluted with 800 ll ofan ethanol: acetone solution (80:20 v/v), placed in 96well plates and the optical density was measured at590 nm using the TECAN Infinite 200 microplate

reader. Three independent experiments were per-formed in duplicate. Biofilm formation by eachLactobacillus strain was calculated by subtracting thenegative control value (medium without bacteria)from the mean value of each sample. Strains werecategorized as biofilm forming when absorbance was �2times higher than the negative control value (Stepanovi�cet al. 2007).

Biofilm formation assay on AGS cells

Sterile circular glass coverslips were placed in 24 well-flat bottom plates, and treated with 350 ll of poly-L-lysine (1mg ml�1, Merck) for ensuring better cellattachment to glass. After 2 h, poly-L-lysine wasremoved and plates were washed twice with PBS.AGS cells were seeded (5� 104 cells well�1) in com-plete DMEM medium and incubated at 37 �C, 5%CO2 atmosphere for 24 h. Then, AGS cells were care-fully washed three times with PBS and bacterial sus-pensions (1.5� 108 or 1.5� 109 cells ml�1) wereadded with fresh DMEM medium supplemented with10% of FBS and without antibiotics. The lactobacilliand AGS cell co-culture system was incubated for 0,4, 8, 12, 24 and 48 h. After the incubation time, non-attached bacteria and supernatant medium were dis-carded. Coverslips were carefully washed with PBSand fixed with 2.5% glutaraldehyde for 48 h at 4 �C.Preparations were then washed with PBS and dehy-drated with ethanol gradient and dried (Salas-Jaraet al. 2016). Lactobacillus biofilm formation on AGScells was visualized using an Auto-scan model U1scanning electron microscope (ETEC Corporation) atthe Center for Microscopy and Spectroscopy,University of Concepcion (Chile).

Lactobacillus adhesion on AGS cells

As described above, cells were grown on coverslipsand Lactobacillus strains were added for differentincubation times. Then, cells were washed threetimes with PBS and treated with 0.25% Trypsin2mM EDTA 1� (Corning) to detach cells fromglass surface. Then, cells were suspended in 700 llof saline solution as previously described (Salas-Jaraet al. 2016). Suspensions were vortexed and 10-folddilutions were made. Finally, aliquots of dilutedsamples were plated by triplicate in MRS agarplates for counting by microdrop technique(Herbert 1990).

BIOFOULING 3

AGS and THP-1 cells viability and cytotoxicity

The cytotoxic effect of the different treatments on AGSand THP-1 cells was evaluated by the determination oflactate dehydrogenase (LDH) in the culture supernatantand was expressed as Units l�1 by measuring the forma-tion of the reduced form of nicotinamide adeninedinucleotide (NAD) using the Wiener reagents andprocedures (Wiener Lab, Buenos Aires, Argentina).

In addition, cell viability was evaluated with theBromide reduction assay of 3 (4,5-dimethyl-2-thiazoyl)-2,5-diphenyltetrazole (MTT) ROCHE Cell ProliferationKit I (MTT) following the manufacturer’s instructions.Cells were cultured in 96 well plate, incubated for 24 hand stimulated with different doses of bacterial suspen-sions. After incubation time, cells were washed threetimes with PBS and MTT was added for 4 h. The result-ing formazan salts were dissolved with solubilizationsolution and measured colorimetrically at 560 nm.

Cytokine profiles

The levels of TNF-a, IL-1b, IL-6, CXCL8 (IL-8), andCCL2 (MCP-1) (pg ml�1) were determined in AGScell culture supernatant. The levels of TNF-a, IL-1b,IL-6, IL-8, IL-10 and IFN-c (pg ml�1) were deter-mined in THP-1 macrophages. Cell culture superna-tants were kept in �80 �C until ELISA wereperformed. Cytokines and chemokines levels weremeasured with commercial enzyme-linked immuno-sorbent assay (ELISA) kits following the manufac-turer’s recommendations (DuoSet R&D Systems).Tests were performed in triplicate. The valuesreported correspond to the average of the determina-tions ± standard deviation.

H. pylori adhesion to AGS cells

In order to evaluate the effect of L. rhamnosus UCO-25A on the adhesion of H. pylori, pre-incubation andco-incubation experiments were performed. AGS wereseeded in 24 well plates and grown for 24 h. Differentdoses of the lactic acid bacterium strain (105 and 107

CFU ml�1) were added; for pre-incubation experi-ments AGS cells were stimulated with L. rhamnosusUCO-25A for 12 h. After that period of time, cellswere washed to eliminate non-adherent bacteria andchallenged with H. pylori. In co-incubation assays lac-tobacilli and H. pylori were administered simultan-eously. For both conditions, AGS cells werechallenged with 6� 107 CFU ml�1 of H. pylori ml�1

for 24 h. After the incubation period, cells werewashed three times with PBS, trypsinized, and serial

dilutions were prepared in PBS to determine H. pyloricell counts. Dilutions were seeded in triplicate onColumbia agar plates. Infected AGS cells without pro-biotic treatment were considered as 100% adhesion.

Statistical analysis

Experiments were performed in triplicate and theresults expressed as the means ± SD. For the compari-son of two groups the Student’s t-test was used andfor the comparison of more than two groups a one-way analysis of variance (ANOVA) were performed.Fisher test was used for posteriori comparisons. In allcases a level of significance of 0.05 was used.

Results

L. rhamnosus UCO-25A forms biofilms in abioticand biotic surfaces

The first aim was to determine whether L. rhamnosusUCO-25A was able to form biofilms in abiotic surfa-ces. Considering that the well-characterized probioticstrain L. rhamnosus GG is able to form biofilms(Lebeer et al. 2007), this probiotic strain of the samespecies was used for comparisons. Both, UCO-25Aand GG strains were able to form biofilms on nega-tively charged glass surfaces after initial inoculums of1.5� 108 (Figure 1A) as well as 1.5� 109 bacterialcells (Figure 1B). When the biofilm formation abilitywas compared using different culture media, thestrains showed subtle differences in absorbance values.L. rhamnosus UCO-25A showed a higher capacity forbiofilm formation in BHI plus glucose medium at 8 hwhen the lowest inoculum was used (Figure 1A).When MRS and DMEM media were evaluated withan inoculum of 1.5� 108 cells ml�1, there were nosignificant differences among the strains (Figure 1A).Stable OD590 values were observed in MRS plus glu-cose medium between hours 4 to 48 for both strains,and poor biofilm formation was obtained withDMEM plus FBS media, with a peak at 12 h (Figure1A). When a higher bacterial inoculum was used(Figure 1B), the absorbance values were significantlydifferent at 12, 24 and 48 h in BHI plus glucose broth,suggesting a higher biofilm formation ability for L.rhamnosus UCO-25A in BHI plus glucose medium at12 h. Conversely, when MRS was used, L. rhamnosusGG exhibited greater biofilm formation at 12 h(Figure 1B). The inoculum of 1.5� 109 bacterialcells ml�1 showed stable OD590 values in MRS plusglucose after reaching a peak at 8 h (Figure 1B). Forboth inocula, when BHI plus glucose broth or DMEM

4 V. GARCIA-CASTILLO ET AL.

plus FBS were used, biofilm formation decreased sig-nificantly after a peak reached between 8 and 12 h(Figure 1A and B). L. rhamnosus UCO-25A and L.rhamnosus GG biofilm density in MRS plus glucosewas stable after reaching a peak around 8 h up to48 h. When the lactobacilli strains were grown in BHIplus glucose broth or in DMEM plus FBS, after apeak reached around 8–12 h, biofilm formationdecreased significantly (Figure 1).

In addition, L. rhamnosus UCO-25A and L. rham-nosus GG biofilm formation on AGS cells was eval-uated by SEM imaging at different incubation timesusing MOIs of 1,000:1 and 10,000:1 (1.5� 108 and1.5� 109 bacterial cells as initial inocula, respectively)in DMEM plus FBS. These high bacterial inocula arenecessary to demonstrate biofilm formation sincelower MOIs require prolonged incubation times thatexceed the viability of the cell cultures. As shown inFigure 2, few bacterial cells were attached to AGScells when MOI 1,000:1 was used as the initial inocu-lum. Mild microcolony formation was observed from4 to 12 h for both bacterial strains as shown in Figure2A. Denser biofilm structures were formed from 4 hwhen MOI 10,000:1 was used as the initial inoculum(Figure 2B). L. rhamnosus GG biofilm structuredecreased after 24 h when the lower initial inoculumwas used. On the contrary, more adhered bacteria

were found in AGS cells at 48 h when cells weretreated with L. rhamnosus UCO-25A (Figure 2A).Lactobacilli seemed to form aggregates, comparedwith rod bacteria adhered to AGS at 0 to 4 h. Whenthe MOI 10,000:1 was evaluated both L. rhamnosusGG and UCO-25A showed a denser biofilm structurefrom 8h onwards (Figure 2B). Bacterial counts wereperformed after each incubation time to assess theviable lactobacilli adhered to AGS cells. As observedin Figure 2C, the lower initial inoculum reached apeak at 8 h and maintained stable up to 48 h for bothstrains. Similarly, UCO-25A and GG bacterial countsfor the higher initial inoculum showed a peakbetween 8 and 12 h. However, both strains showed adecrease in bacterial cell counts after 12 h.

L. rhamnosus UCO-25A exert immunomodulatoryeffects on AGS and THP-1 cells

In the previous experiments, high MOIs were used inorder to clearly demonstrate the ability of UCO-25Astrain to form biofilms on AGS cells. However, itshould be considered that those MOIs are of a muchhigher possible bacterial density in the stomach.Then, for the evaluation of the immunomodulatoryeffects of L. rhamnosus UCO-25A MOIs of 1:1 and100:1 (1.5� 105 and 1.5� 107 bacterial cells as initial

Figure 1. Biofilm formation on abiotic surfaces by L. rhamnosus UCO-25A and L. rhamnosus GG. Biofilm formation was evaluatedon glass surfaces using initial inoculums of (A) 1.5� 108 CFU ml�1, or (B) 1.5� 109 CFU ml�1; white bars correspond to L. rham-nosus UCO-25A, black bars to L. rhamnosus GG. Different culture media were evaluated including BHI plus 2% glucose, MRS plus2% glucose, or DMEM plus 10% FBS. Biofilm formation was assessed by the crystal violet method after incubation for 0, 4, 8, 12,24 and 48 h. The results represent data from three independent experiments per duplicate. Results are expressed as means± SD.�� Indicates significant differences (p< 0.05) applying a Student’s t-test.

BIOFOULING 5

inocula, respectively) were used. First, it eas deter-mined whether L. rhamnosus UCO-25A producedadverse effects on AGS cells. Cellular damage and cellviability were assessed by the LDH and MTT assays,respectively. Cells without treatment were used as con-trols. As shown in Figure 3A, there were no significantdifferences between lactobacilli-treated AGS cells anduntreated controls. Conversely, when cellular damageand cell viability was evaluated on differentiated THP-1 macrophages, a slightl reduction in viability as wellas an increase in LDH release was observed in lactoba-cilli-treated macrophages when compared withuntreated controls (Figure 3B). Therefore, L. rhamno-sus UCO-25A did not exert a substantial effect on theviability of both AGS and THP-1 cells since 85–100%of cells were viable in all cases.

Next, the effect of L. rhamnosus UCO-25A on thecytokine profile of AGS and THP-1 after incubationfor 12 h was evaluated. As shown in Figure 4A, theproduction of pro-inflammatory cytokines TNF-a, IL-1b, IL-6 as well as the chemokines MCP-1/CCL2 andCXCL-8/IL-8 was increased in AGS treated with L.rhamnosus UCO-25A when compared with untreatedcells. There was no significant difference in the stimu-latory response among bacterial doses evaluated.

Similarly, TNF-a, IL-6, IFN-c and IL-10 levelswere measured in macrophage- differentiated THP-1macrophages supernatants after incubation for 12 hwith the UCO-25A strain (Figure 4B). The levels ofthe pro-inflammatory factors TNF-a, IL-6, IL-8 andIFN-c were significantly enhanced in THP-1 macro-phages stimulated with L. rhamnosus UCO-25A. Inaddition, the immunomodulatory cytokine IL-10 wasalso significantly increased in differentiated THP-1macrophages treated with L. rhamnosus UCO-25Awhen compared with untreated cells (Figure 4B).

L. rhamnosus UCO-25A differentially modulatesthe inflammatory response triggered by H. pyloriin AGS cells

In order to evaluate the potential protective effect ofthe UCO-25A strain on H. pylori infection, the abilityof lactobacilli to reduce the adhesion of the pathogenon AGS cells was first studied (Figure 5). In pre-incu-bation experiments, AGS cells were treated with L.rhamnosus UCO-25A for 12 h, washed for the elimin-ation of non-adherent bacteria and then challengedwith the pathogen. On the other hand, co-incubationassays consisted in culturing AGS cells with

Figure 2. Biofilm formation on human gastric epithelial cells (AGS) by L. rhamnosus UCO-25A and L. rhamnosus GG evaluated bySEM imaging. (A) Biofilm formation on using 1.5� 108 CFU ml�1 inoculum of L. rhamnosus UCO-25A (upper image) and L. rham-nosus GG (lower image) evaluated by SEM imaging after incubation for 0, 4, 8, 12, 24 and 48 h. Yellow arrows show L. rhamnosusadhesion at 0 h. The scale bars correspond to 5lm (magnification: 5,000�). (B) Biofilm formation on AGS cells using 1.5� 109

CFU ml�1 inoculum of L. rhamnosus UCO-25A (upper image) and L. rhamnosus GG (lower image) evaluated by SEM imaging afterincubation for 0, 4, 8, 12, 24 and 48 h. Yellow arrows show L. rhamnosus GG adhesion at 0 h. The scale bars correspond to 5lm(magnification: 5,000�). (C) Lactobacillus adhesion using initial inocula of 1.5� 108 CFU ml�1 (dots), or 1.5� 109 CFU ml�1

(squares) was evaluated by plate counts after different incubation times. The results represent data from three independentexperiments. �� Indicates significant differences (p< 0.05) applying a Student’s t-test.

6 V. GARCIA-CASTILLO ET AL.

lactobacilli and H. pylori simultaneously. As observedin Figure 5, in both sets of experiments, the UCO-25A strain significantly reduced the adhesion of thegastric pathogen to AGS cells. However, the bestinhibitory results were obtained with the prophylactictreatment since H. pylori adhesion was reduced by50% compared to the 64% found in co-incubationexperiments. No significant differences were observedbetween the two evaluated doses (Figure 5).

Pre-incubation experiments were selected for cyto-kines profile determination. As shown in Figure 6A,the challenge of AGS cells with the gastric pathogen

significantly increased cellular damage and reducedcell viability as demonstrated by LDH and MTTassays, respectively. Interestingly, the prophylactictreatment with the UCO-25A strain improved viabil-ity and reduced cellular damage of H. pylori infectedcells (Figure 6A).

The ability of L. rhamnosus UCO-25A to modu-late the production of cytokines and chemokinesduring H. pylori infection was assessed by determin-ing the levels of TNF-a, IL-1b, IL-6, IL-8/CXCL8and MCP-1/CCL-2 in culture supernatants (Figure6B). The challenge of AGS cells with the gastric

Figure 3. The effect of L. rhamnosus UCO-25A on the viability and cytotoxicity of (A) human gastric epithelial cells (AGS cells) and(B) human macrophages (THP-1 cells). AGS cells and Phorbol 12-Myristate 13-Acetate (PMA)-differentiated THP-1 macrophageswere treated with different doses of L. rhamnosus UCO-25A. The percentage of viable cells by the MTT colorimetric assay and thelevels of LDH in culture supernatants were determined after lactobacilli stimulation for 12 h. The results represent data from threeindependent experiments. Results are expressed as means± SD. a Means with different letters differ significantly (p< 0.05).

BIOFOULING 7

pathogen significantly increased the levels of all thepro-inflammatory factors evaluated. In addition, itwas observed that lactobacilli-treated AGS cells sig-nificantly decreased the levels of the inflammatorycytokines TNF-a and IL-1b as well as the chemo-kines MCP-1 and IL-8 when compared to infected

controls (Figure 6B). On the contrary, IL-6 produc-tion was increased in L. rhamnosus UCO-25A-treatedAGS cells. No significant differences were observedbetween the two evaluated doses in the capacity tomodulate cytokines and chemokines production(Figure 6B).

Figure 5. The effect of L. rhamnosus UCO-25A on adhesion of H. pylori to human gastric epithelial cells (AGS cells). Pre-incubationand co-incubation experiments were performed with low doses of the probiotic strain (105 or 107cells ml�1). In pre-incubationexperiments AGS cells were stimulated with L. rhamnosus UCO-25A for 12 h, washed for the elimination of non-adherent bacteriaand challenged with H. pylori. In co-incubation assays lactobacilli and H. pylori were administered simultaneously. For both condi-tions, AGS cells were then challenged with H. pylori for 24 h. The results represent data from three independent experiments.Results are expressed as means± SD. a,b Means with different letters differ significantly (p< 0.05).

Figure 4. The effect of L. rhamnosus UCO-25A on cytokine production by (A) human gastric epithelial cells (AGS cells) and (B)human macrophages (THP-1 cells). Cells were treated with different doses of L. rhamnosus UCO-25A. The levels of TNF-a, IL-1b, IL-6, IL-8 and MCP-1 (pg ml�1) were determined in AGS culture supernatants after lactobacilli stimulation for 12 h. The levels ofTNF-a, IL-6, IL-8, IL-10 and IFN-c (pg ml�1) in culture supernatants were determined in Phorbol 12-Myristate 13-Acetate (PMA)-dif-ferentiated THP-1 macrophages after lactobacilli stimulation for 12 h. The results represent data from three independent experi-ments. Results are expressed as means± SD. a,b Means with different letters differ significantly (p< 0.05).

8 V. GARCIA-CASTILLO ET AL.

Figure 6. The effect of L. rhamnosus UCO-25A on the viability, cytotoxicity and cytokine production of human gastric epithelialcells (AGS cells) after the challenge with H. pylori. AGS cells were treated with different doses of L. rhamnosus UCO-25A for 12 hand then challenged with the gastric pathogen. (A) The percentage of viable cells by MTT colorimetric assay and the levels ofLDH in culture supernatants were determined 24 h after H. pylori infection. (B) The levels of TNF-a, IL-1b, IL-6, IL-8 and MCP-1 (pgml�1) in culture supernatants were determined 24 h after H. pylori infection. The results represent data from three independentexperiments. Results are expressed as means± SD. a–c Means with different letters differ significantly (p< 0.05).

BIOFOULING 9

L. rhamnosus UCO-25A differentially modulatesthe inflammatory response triggered by H. pyloriin THP-1 cells

Next, he ability of L. rhamnosus UCO-25A to modu-late the production of cytokines and chemokines byTHP-1 macrophages after H. pylori or LPS challengeswas evaluated. As shown in Figure 7A, both H. pyloriinfection and the LPS challenge significantlydecreased the viability and increased cellular damage

of differentiated THP-1 macrophages. Although bothpro-inflammatory treatments had deleterious effectson THP-1 cells, the alterations induced by LPS weregreater than those induced by the gastric pathogen.Of interest, the UCO-25A strain was able to signifi-cantly reduce the damage induced by both H. pylori-and LPS in THP-1 cells (Figure 7A).

Considering that in the previous determinationsthere were no significant differences between the twodoses of L. rhamnosus UCO-25A, only the higher

Figure 7. The effect of L. rhamnosus UCO-25A on the viability, cytotoxicity and cytokine production by human macrophages(THP-1 cells) after the challenge with H. pylori or LPS. Phorbol 12-Myristate 13-Acetate (PMA)-differentiated THP-1 macrophageswere treated with different doses of L. rhamnosus UCO-25A for 24 h and then challenged with the gastric pathogen or LPS. (A)The percentage of viable cells by MTT colorimetric assay and the levels of LDH in culture supernatants were determined 12 h afterH. pylori infection or LPS stimulation. (B) PMA-differentiated THP-1 macrophages were treated with 107 cells of L. rhamnosus UCO-25A for 24 h and then challenged with the gastric pathogen or LPS. The levels of TNF-a, IL-6, IL-8, IL-10 and IFN-c (pg ml�1) inculture supernatants were determined 24 h after H. pylori infection or LPS stimulation. The results represent data from three inde-pendent experiments. Results are expressed as means± SD. a–d Means with different letters differ significantly (p< 0.05).

10 V. GARCIA-CASTILLO ET AL.

dose was used for the analysis of cytokine production(Figure 7B). Both H. pylori as well as the LPS chal-lenges increased the production of the inflammatoryfactors TNF-a, IL-6, IL-8 and IFN-c by differentiatedTHP-1 macrophages. However, LPS exerted a higherability to enhance the production of those inflamma-tory factors when compared with H. pylori (Figure7B). L. rhamnosus UCO-25A treated THP-1 cells hadsignificantly lower levels of TNF-a and IL-8 produc-tion after H. pylori infection or LPS challenge, whilethe levels of IFN-c were significantly higher. No evi-dent effect was observed in IL-6 levels when UCO-25A treated THP-1 cells when compared to controlsin H. pylori challenge experiments. On the otherhand, IL-6 levels were improved in LPS-challengedcells treated with the UCO-25A strain when com-pared to controls (Figure 7B).

Both H. pylori and LPS increased the productionof the immunoregulatory cytokine IL-10 by differenti-ated THP-1 macrophages. In addition, the productionof IL-10 was significantly increased by L. rhamnosusUCO-25A in the two sets of experiments (Figure 7B).

L. rhamnosus UCO-25A biofilm state improveresistance to H. pylori

Finally, the ability of both lactobacilli biofilms toreduce the adhesion of H. pylori to AGS cells (Figure8) was evaluated. AGS cells were treated with1.5� 108 cells ml�1 (MOI 1000:1) of L. rhamnosusUCO-25A or GG for 24 hs in order to favor biofilmformation and then cells were challenged with thegastric pathogen. Both Lactobacillus strains signifi-cantly reduced the adhesion of H. pylori to AGS cells.

In addition, L. rhamnosus UCO-25A and L. rhamno-sus GG were able to significantly reduce the produc-tion of both CXCL8/IL-8 and CCL2/MCP-1 by AGScells after H. pylori infection (Figure 8). Of interest,the percentage of inhibition reached by L. rhamnosusUCO-25A biofilm showed a tendency to higher valuesthan the observed for planktonic cells (Figure 5), aswell as the reduction in inflammatory chemokines(Figure 6).

Discussion

Culture independent techniques and accessible high-throughput sequencing technologies have contributedto reveal the role of commensal bacteria in health anddisease (Walker and Talley 2014). However, comparedwith intestinal microbiota, gastric microbial compos-ition and its impact on health have been lessexplored. Recently, in a study performed by Klymiuket al. (2017) the characteristics of the murine gastricmicrobiota and its relation with H. pylori infectionwas analyzed. Comparative analysis revealed numer-ous significant abundance differences from phylum tospecies level between control and H. pylori-infectedmice. The infection with the gastric pathogen consist-ently affected the family Lactobacillaceae in mice ofdifferent ages. In line with these findings, Garc�ıa et al.(2012) reported that the co-existence of bothLactobacillus spp. and H. pylori is low in the humangastric mucosa of symptomatic patients. Moreover, areduced abundance of Lactobacillales was found inthe gastric fluids of H. pylori-infected urban children(Brawner et al. 2017). The qualitative alterations inthe gastric microbiota in persons infected with H.

Figure 8. The effect of L. rhamnosus biofilms on adhesion of H. pylori and cytokine production by human gastric epithelial cells(AGS cells). AGS cells were stimulated with L. rhamnosus UCO-25A or L. rhamnosus GG for 24 h, washed for the elimination ofnon-adherent bacteria and challenged with H. pylori for 24 h. The levels of IL-8, and MCP-1 (pg ml�1) in culture supernatantswere determined 24 h after H. pylori infection. The results represent data from three independent experiments. Results areexpressed as means± SD. a,b Means with different letters differ significantly (p< 0.05).

BIOFOULING 11

pylori have been implicated in the development ofgastric ulcers and cancer (Maldonado-Contreras et al.2011; Coker et al. 2017) and therefore, the manipula-tion of the gastric microbiota has emerged as aninteresting alternative to reduce the severity of H.pylori-mediated diseases.

Research in cellular and animal models as well asclinical trials support that various species ofLactobacillus may have a beneficial impact on H.pylori eradication when used as adjuvants or mono-therapy (Homan and Orel 2015; Malfertheiner et al.2017; Losurdo et al. 2018). The ability of probioticlactobacilli to produce antimicrobial compounds,competitively inhibit the adhesion to gastric epithelialcells or modulate the immune system have been asso-ciated with those beneficial effects (Homan and Orel2015; Goderska et al. 2017). However, it should benoted that the majority of those studies were per-formed with lactobacilli from different origins andonly few of them evaluated strains originally isolatedfrom the human gastric mucosa.

Taking into consideration that bacteria originallyisolated from the human stomach would be more effi-cient in colonizing the gastric mucosa and therefore,in inhibiting the adhesion and growth of H. pylori,the authors have previously isolated several lactic acidbacteria strain from human gastric tissue in order toevaluate them as potential probiotic strains (Garciaet al. 2009). Among the strains isolated, L. rhamnosusUCO-25A excelled for its ability to survive gastro-intestinal conditions. Here, it was found that thisstrain could be used as an anti-H. pylori probioticstrain as it was demonstrated that it is capable ofinhibiting the adhesion of the gastric pathogen to epi-thelial cells. Moreover, it was also demonstrated thatL. rhamnosus UCO-25A is able to differentiallymodulate the inflammatory response triggered by H.pylori infection in both non-immune and immunecells. The experiments on AGS cells and differentiatedTHP-1 macrophages clearly showed that the UCO-25A strain exerted an immunomodulatory effect aftera H. pylori challenge. These findings are of import-ance since it is known that H. pylori infection causean aggressive pro-inflammatory immune response inthe gastric tissue that is associated to the severity andprogression of the diseases caused by this pathogen(Posselt et al. 2013).

Transcriptomic studies performed during infection-challenge experiments in mice revealed that H. pylorisignificantly alters gene expression in the stomach ofinfected mice when compared to controls (Klymiuket al. 2017). A significant increase in the expression of

inflammatory factors was found in H. pylori-infectedmice that correlated with an enhanced mononuclearand neutrophilic infiltrates in the lamina propria ofthe gastric tissue. Moreover, inflammation mediatedsevere gastric tissue damage. It was also observed thatH. pylori infection in mice increased the levels ofCXCL1, the functional homolog of IL-8, as well asTNF-a, IL-6 and IL-1b in gastric tissues (Kumar et al.2002; McNamee et al. 2011). In vitro studies haveshown that both non-immune and immune cells par-ticipate in the inflammatory response against this gas-tric pathogen. Transcriptomic studies performed byEftang et al. (2012) in AGS cells challenged with H.pylori indicated that the gastric pathogen is able tosubstantially increase the expression of IL-8 in epithe-lial cells among thousands of genes evaluated. Ofinterest, it was reported that the expression levels ofIL-8 in the stomach of H. pylori-infected patients cor-relate with the severity of gastritis (Xuan et al. 2005)and the risk of gastric cancer (Brandt et al. 2005;Yamada et al. 2013). On the other hand, it was shownthat macrophages play key roles in the inflammatorydisease caused by this gastric pathogen. Studies inhuman and mouse macrophages have demonstratedthat H. pylori efficiently triggers the production ofTNF-a, MCP-1, IL-8, IL-6 and IFN-c by these cells(Fehlings et al. 2012; Ansari et al. 2014; Kameokaet al. 2016; Wang et al. 2017).

In line with these previous works, it was observedhere that the challenge of AGS cells or PMA-differen-tiated THP-1 macrophages with H. pylori enhancedthe production of inflammatory cytokines and chemo-kines. The challenge of AGS cells with the gastricpathogen significantly increased the levels of TNF-a,IL-1b, IL-6, IL-8 and MCP-1 while the levels of TNF-a, IL-8, IL-6 and IFN-c were enhanced in macro-phages. Interestingly, L. rhamnosus UCO-25A reducedthe production of TNF-a, IL-1b, IL-8 and MCP-1 inAGS cells and TNF-a and IL-8 in macrophages afterH. pylori infection, indicating a substantial anti-inflammatory effect. These findings are of value sinceseveral studies have shown that the reduction ininflammatory factors production by gastric epithelialcells and macrophages, especially IL-8 and TNF-a, areassociated with a less severe infection and sequels(Brandt et al. 2005; Xuan et al. 2005; Posselt et al.2013; Yamada et al. 2013). In addition to its ability toreduce inflammatory factors in epithelial cells andmacrophages, L. rhamnosus UCO-25A was also cap-able of enhancing the production of the immunoregu-latory cytokine IL-10 by macrophages. Consideringthat an appropriate balance between inflammatory

12 V. GARCIA-CASTILLO ET AL.

and anti-inflammatory factors is necessary to elimin-ate the pathogen, avoid chronic infection and evadeinflammatory tissue damage, the increase in IL-10induced by the UCO-25A strain could be also favor-able during H. pylori infection. However, it should beconsidered that some reports indicate that excessiveIL-10 production might help the pathogen to persistin the gastric mucosa and to contribute to the devel-opment of chronic diseases (Sato et al. 2019).Therefore, detailed in vivo kinetics studies of theeffect of L. rhamnosus UCO-25A on gastric cytokinesproduction in the context of H. pylori infection arenecessary in order to clearly demonstrate the potentialbeneficial effect of this probiotic strain.

It should be noted that the immunoregulatoryeffect of L. rhamnosus UCO-25A was not completelyanti-inflammatory since the levels of IL-6 and IFN-cafter H. pylori challenges were significantly increasedin AGS and THP-1 cells respectively, when comparedto cells that were not in contact with lactobacilli. Asmentioned previously, several studies have shown thatH. pylori infection is associated with increased levelsIL-8 and IL-6 within the gastric mucosa. Interestingly,the mechanisms of H. pylori-mediated IL-6 inductiondiffered from those involved in the induction of IL-8,and therefore the production of both cytokines isindependent of each other in gastric epithelial cells(Yamaoka et al. 2004; Lu et al. 2005). In addition,although a chronic production of IL-6 has been asso-ciated with the development of gastric cancer(Kinoshita et al. 2013), this cytokine has importantprotective effects in the earliest phases of the infec-tion. IL-6 is pleiotropic cytokine with a range of dif-ferent functions. One role of IL-6 is to improveresistance to apoptosis that promotes cell viability(Yao et al. 2010). It has been reported that gastric epi-thelial cells increase the production of this cytokine inresponse to stress factors in order to improve theirsurvival (Marcus et al. 2013). In addition, IL-6 alsostimulates the T cell responses that mediate protectionagainst infections. On the other hand, studies haverevealed that H. pylori has a facultative intracellularbacterial behavior and therefore it invades not onlyepithelial cells, but also macrophages (Fehlings et al.2012). It has been demonstrated that H. pylori is ableto manipulate autophagic mechanisms (Wang et al.2009) and IFN-c production (Shan et al. 2015) to rep-licate and survive within macrophages. Moreover,experiments in IFN-c knock-out mice showed thatanimals fail to develop gastric inflammation but haveincreased susceptibility to H. pylori colonization (Peeket al. 2010). Then, the ability of L. rhamnosus UCO-

25A to improve IL-6 and IFN-c could have a role inthe protection against H. pylori infection that deservesdeeper in vivo research.

Interestingly, the ability of L. rhamnosus UCO-25Ato protect against H. pylori infection and modulatethe inflammatory response in AGS cells showed a ten-dency to be improved when lactobacilli were allowedto form a biofilm on gastric cells. It was establishedthat adhesion and biofilm formation are strain spe-cific properties that reportedly contribute to the per-manence of lactobacilli in mucosal tissues (Schwabet al. 2014; Leccese Terraf et al. 2016). Moreover, bio-film formation in beneficial Lactobacillus strains isnow considered as a valuable property that can favorbacterial colonization when a probiotic product isadministered to the host (Lebeer et al. 2007; LecceseTerraf et al. 2016; Olson et al. 2016). Indeed, similarto the present work, some studies have reported thatbeneficial effects of probiotic lactobacilli could beenhanced by the induction of biofilm formation. In arecent study, Olson et al. (2018) evaluated whetherthe improvement in biofilm formation by a probioticL. reuteri strain induced by the addition of biofilm-promoting substances was able to enhance its abilityto prevent the development of necrotizing enterocoli-tis in an animal model. Of note, the administration ofa single dose of L. reuteri in its biofilm state signifi-cantly reduced intestinal injury and inflammation,and improved host survival.

It should be noted that L. rhamnosus UCO-25Aalso modulated the production of cytokines in theabsence of H. pylori infection. The most noteworthyeffect was its ability to increase IL-6 in epithelial cellsand IFN-c and IL-10 by macrophages. Those changesin the cytokines profiles have been described as typ-ical for several well characterized probiotic strainsincluding L. rhamnosus GG (Lebeer et al. 2007), L.casei CRL431 (Maldonado et al. 2007) and L. rhamno-sus CRL1505 (Salva et al. 2010). Therefore, theseresults open the question whether the UCO-25Astrain could be used to beneficially modulate immun-ity in other mucosal tissues in the context of otherinfections or inflammatory pathologies. This is aninteresting point that the authors intend to study inthe immediate future.

Given the importance of inflammation in H.pylori-mediated diseases, the modulation of theinflammatory response in the gastric mucosa by L.rhamnosus UCO-25A is an attractive alternative forimproving H. pylori eradication and reducing theseverity of the diseases that arise from the resultingchronic inflammation, ranging from dyspepsia and

BIOFOULING 13

gastritis to gastric ulcers and cancer. Further studiesin animal models are necessary to completely charac-terize the immunomodulatory activities of L. rhamno-sus UCO-25A in the context of H. pylori infectionand to evaluate whether planktonic or biofilm formsare the most effective way to exert the beneficialeffects. The present study has demonstrated that lacticacid bacteria that originally colonize the human stom-ach are an interesting source of potential probioticstrains directed to the protection of the gastricmucosa and the inflammatory damage produced byH. pylori.

Disclosure statement

The authors declare that they have no competing interests.

Funding

This study was supported by ANPCyT–FONCyT GrantsPICT-2013-3219 and PICT-2016-0410 to Julio Villena andCONICYT National Doctorate Grant 21150603 to ValeriaGarc�ıa-Castillo. This work was also supported by the JSPSCore-to-Core Program Advanced Research Networks“Establishment of international agricultural immunologyresearch-core for a quantum improvement in food safety”.

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