microorganisms
ArticleLactiplantibacillus plantarum Strains Modulate Intestinal
Innate Immune Response and Increase Resistance toEnterotoxigenic Escherichia coli Infection
Ayelen Baillo 1,† , Julio Villena 2,3, *,† , Leonardo Albarracín 2 , Mikado Tomokiyo 3,4 , Mariano Elean 2 ,Kohtaro Fukuyama 3,4 , Sandra Quilodrán-Vega 5 , Silvina Fadda 1, * and Haruki Kitazawa 3,4, *
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2

3

4

5

*
†

Citation: Baillo, A.; Villena, J.;
Albarracín, L.; Tomokiyo, M.; Elean,M.; Fukuyama, K.; Quilodrán-Vega,
S.; Fadda, S.; Kitazawa, H.
Lactiplantibacillus plantarum Strains
Modulate Intestinal Innate Immune
Response and Increase Resistance toEnterotoxigenic Escherichia coli
Infection. Microorganisms 2023, 11, 63.
https://doi.org/10.3390/
microorganisms11010063
Academic Editor: Harsharn Gill
Received: 30 November 2022
Revised: 19 December 2022
Accepted: 22 December 2022
Published: 25 December 2022

Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons

Laboratory of Technology, Reference Centre for Lactobacilli (CERELA-CONICET),
Tucuman CP4000, Argentina
Laboratory of Immunobiotechnology, Reference Centre for Lactobacilli (CERELA-CONICET),
Tucuman CP4000, Argentina
Food and Feed Immunology Group, Laboratory of Animal Food Function, Graduate School of Agricultural
Science, Tohoku University, Sendai 981-8555, Japan
Livestock Immunology Unit, International Education and Research Center for Food and Agricultural
Immunology (CFAI), Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan
Laboratory of Food Microbiology, Faculty of Veterinary Sciences, University of Concepción,
Chillán 3820572, Chile
Correspondence: jcvillena@cerela.org.ar (J.V.); sfadda@cerela.org.ar (S.F.);
haruki.kitazawa.c7@tohoku.ac.jp (H.K.)
These authors contributed equally to this work.

Abstract: Currently, probiotic bacteria with not transferable antibiotic resistance represent a sustainable strategy for the treatment and prevention of enterotoxigenic Escherichia coli (ETEC) in farmanimals. Lactiplantibacillus plantarum is among the most versatile species used in the food industry,either as starter cultures or probiotics. In the present work, the immunobiotic potential of L. plantarum
CRL681 and CRL1506 was studied to evaluate their capability to improve the resistance to ETEC infection. In vitro studies using porcine intestinal epithelial (PIE) cells and in vivo experiments in mice
were undertaken. Expression analysis indicated that both strains were able to trigger IL-6 and IL-8
expression in PIE cells in steady-state conditions. Furthermore, mice orally treated with these strains
had significantly improved levels of IFN-γ and TNF-α in the intestine as well as enhanced activityof peritoneal macrophages. The ability of CRL681 and CRL1506 to beneficially modulate intestinal
immunity was further evidenced in ETEC-challenge experiments. In vitro, the CRL1506 and CRL681
strains modulated the expression of inflammatory cytokines (IL-6) and chemokines (IL-8, CCL2,CXCL5 and CXCL9) in ETEC-stimulated PIE cells. In vivo experiments demonstrated the ability ofboth strains to beneficially regulate the immune response against this pathogen. Moreover, the oral
treatment of mice with lactic acid bacteria (LAB) strains significantly reduced ETEC counts in jejunum
and ileum and prevented the spread of the pathogen to the spleen and liver. Additionally, LAB
treated-mice had improved levels of intestinal IL-10 both at steady state and after the challenge with
ETEC. The protective effect against ETEC infection was not observed for the non-immunomodulatory
TL2677 strain. Furthermore, the study showed that L. plantarum CRL1506 was more efficient thanthe CRL681 strain to modulate mucosal immunity highlighting the strain specific character of thisprobiotic activity. Our results suggest that the improved intestinal epithelial defenses and innate
immunity induced by L. plantarum CRL1506 and CRL681 would increase the clearance of ETEC and at
the same time, protect the host against detrimental inflammation. These constitute valuable features
for future probiotic products able to improve the resistance to ETEC infection.
Keywords: probiotics; lactic acid bacteria; Lactiplantibacillus plantarum; intestinal immune response;
enterotoxigenic Escherichia coli

Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Microorganisms 2023, 11, 63. https://doi.org/10.3390/microorganisms11010063

https://www.mdpi.com/journal/microorganisms

Microorganisms 2023, 11, 63

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1. Introduction
Enterotoxigenic Escherichia coli (ETEC) is one of the most common causes of human diarrhea in developing countries [1] These bacteria can also affect farm animals. For example,
it is the main bacterial etiologic agent of post-weaning diarrhea in pigs [2]. ETEC-associateddisease causes huge economic losses in the global swine industry due to high morbidity
and mortality, substantial veterinary costs, and stunted growth of animals [3]. Symptomsof ETEC infection include watery diarrhea with associated depression, loss of appetite,and dehydration. ETEC strains adhere to small intestinal epithelial cells (IECs) throughflexible fimbriae present on their surface, which mediate recognition and adherence to thecorresponding receptors [4]. ETECs expressing F4 fimbriae are the most prevalent strainsin pigs [3]. After colonization, porcine ETEC strains produce one or more thermolabile (LT)and/or thermostable enterotoxins (ST) [2], which activate a flow of electrolytes towards theintestinal lumen, creating a hypertonic environment. Consequently, water moves from theepithelial cells into the intestinal lumen causing hypersecretory diarrhea. In addition toenterotoxins damage, the lipopolysaccharide (LPS) from the cell wall can induce intestinal
damage through the stimulation of the inflammatory response [5]. The innate immune
response of IECs is initiated when the pathogen-associated molecular pattern (PAMPs)
such as LPS, binds to specialized pattern recognition receptors, including membrane-boundToll-like receptors (TLRs). This interaction activates the signaling pathway for nuclear
factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) [6], leading to thetranscriptional expression of various pro-inflammatory chemokines, cytokines, and antimicrobial peptides that trigger the recruitment and activation of inflammatory cells [7,8]. The
TLR4-mediated production of inflammatory cytokines (TNF-α, IL-6, IL-17), chemokines
chemoattractant for neutrophils (IL-8, CXCL5), monocytes (CCL2, CCL8) and lymphocytes(CXCL9, CXCL10, CXCL11) can contribute to intestinal tissue damage in pigs during ETEC
infection if it is not regulated properly [5,9].
Currently, the treatment and prevention of ETEC in animals are based on the use of
antimicrobials. This has led to the emergence of antibiotic resistant bacterial strains aroundthe world [10]. Emerging clones carry multiple resistance genes, have high dissemination
capacity and high pathogenicity [11]. Therefore, it is necessary to develop alternatives that
help to reduce the impact of ETEC infection and eliminate the need for antimicrobial treatment. In this sense, probiotics represent an attractive strategy since they are selected frombacteria with not transferable antibiotic resistance, and they do not have the deleterious
effect of antibiotics on the intestinal microbiota [12]. Research has clearly demonstrated theprotective effects of probiotic microorganisms against pathogenic E. coli. Earlier studies
performed with the probiotic strains Bifidobacterium lactis HN019 [13]. or Lacticaseibacillus
rhamnosus HN001 [14]. demonstrated that their preventive administration to mice significantly improved the resistance against E. coli O157:H7 infection This effect was associatedwith an enhancement of intestinal immunity. Furthermore, by using a piglet model it was
shown that B. lactis HN019 administration increased feed conversion efficiency during
weaning and that this effect was associated with a reduction of the severity of weanling
diarrhea [15]. A multispecies probiotic formulation was also evaluated in its capacity todiminish the severity of post-weaning diarrhea caused by ETEC on newly weaned pigs [16].
The multispecies probiotics improved growth performance by preserving the intestinal
mucosa integrity and diminishing intestinal inflammatory factors like TNF-α. Similarly, theadministration of Pediococcus acidilactici to weaned pigs was shown to increase resistance to
ETEC challenge by modulating the expression of intestinal cytokines [17]. The probiotic
treatment significantly reduced the attachment of ETEC to the intestinal mucosa in pigs anddifferentially regulated the expression of IL-6 and TNF-α. Our research group has experience identifying probiotic strains capable of beneficially regulating the intestinal immune
system, increasing the resistance to ETEC infection [5,18,19]. These immunomodulatory
probiotic strains, referred as immunobiotics, were selected by in vitro assays based on the
porcine intestinal epitheliocyte cell line (PIE cells) developed by our group [20]. In previous
studies, we have shown that TLR4 is strongly expressed in this cell line and that PIE cells

Microorganisms 2023, 11, 63

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can increase the expression of proinflammatory chemokines and cytokines in response
to LPS stimulation [21]. In addition, by using this in vitro model we were able to selectimmunobiotic lactobacilli capable of modulating cytokines and chemokines expression
caused by ETEC or LPS challenges [19,20].
Lactiplantibacillus plantarum can survive in a wide range of environmental niches
including the gastrointestinal tract, easily colonizing the intestine of humans and othermammals [22,23]. In addition, many strains of this bacterial species have shown to possess beneficial properties for the host, including their ability to beneficially modulate theimmune system [24]. Due to these properties, L. plantarum is considered one of the mostwidely used bacterial strains in the food industry both as a starter culture and as a probiotic [25]. Previously, we performed in vitro studies in PIE cells and in vivo studies in micewith different L. plantarum strains and we demonstrated that those lactic acid bacteria (LAB)
possess a differential ability to modulate the respiratory and intestinal innate antiviral
immune responses [26]. Of note, L. plantarum CRL1506 showed a remarkable capacity
to beneficially regulate the mucosal antiviral immune response triggered by the activation of TLR3 [26–28]. The CRL1506 strain improves the production of type I interferons(IFNs) and antiviral factors and differentially regulates the expressions of inflammatory
cytokines and chemokines in epithelial cells from the intestinal [26] and respiratory [28]tracts. Furthermore, in vivo studies in mice demonstrated that the oral treatment with
L. plantarum CRL1506 can modulate the TLR3-mediated intestinal damage through its ability to reduce the expression of IL-15 in the intestinal epithelium and regulate the function of
CD3+ NK1.1+ CD8αα+ intraepithelial lymphocytes [26,29]. Strains like L. plantarum TL2677
do not have those immunomodulatory capacities [26,30]. On the other hand, L. plantarum
CRL681 has a proven technological potential as starter and bioprotective culture for meat andmeat products. The CRL681 strain has remarkable acidogenic [31] and proteolytic [32–34]
activities. In addition. L. plantarum CRL681 has bioprotective potential due to the highinhibitory activity toward Escherichia coli O157:H7 [35].
In the present work we aimed to deepen the characterization of the immunomodulatory properties of L. plantarum CRL1506 and CRL681 particularly focused on their ability
to enhance intestinal immune responses and the resistance against ETEC. Therefore, we
conducted in vitro studies in PIE cells to evaluate their capacity to modulate the innate
immune response on the intestinal mucosa before and after ETEC challenge. In addition,
we performed experiments in mice as preliminary studies to demonstrate in vivo the protective potential of CRL1506 and CRL681 strains against ETEC infection and to provide thescientific basis for carrying out future in vivo studies in pigs.
2. Materials and Methods
2.1. Bacterial Strains and Culture Conditions
Lactiplantibacillus plantarum CRL1506 was originally isolated from goat milk and
CRL681 from fermented sausage. Both strains were obtained from the CERELA culturecollection (Tucumán Argentina). L. plantarum TL2766, originally isolated from human feces,
was included in the experiments as a non-immunomodulatory control strain. The TL2766
strain was obtained from the Meiji dairy culture collection (Tokyo, Japan).
For the experiments of this work, all Lactiplantibacillus strains were activated from frozen
stock and grown on Mann-Rogosa Sharpe Agar (MRS Difco) at 37 ◦ C. After 24 h of incubation,a single colony was transferred to MRS broth (MRS Difco) and was cultured at 37 ◦ C for
24 h. Bacterial cells were then washed three times with phosphate-buffered saline (PBS) andadjusted to appropriate concentrations for in vitro and in vivo experiments using a microscope
and a Petroff-Hausser counting chamber. They were stored at −80 ◦ C until use [21].
Enterotoxigenic Escherichia coli (ETEC) strain 987P (O9: H-: 987 pilus +: heat stabletoxin +) was obtained from the National Institute of Animal Health (Tsukuba, Japan) [21,36].
ETEC cells were cultured on blood agar (5% sheep blood) for 24 h at 37 ◦ C, transferred totryptic soy broth (TSB; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) andcultured 20 h at 37 ◦ C with shaking. After incubation, the bacterial subcultures wereMicroorganisms 2023, 11, 63

4 of 17

centrifuged at 5000× g for 10 min at 4 ◦ C and washed with PBS (pH 7.2). Finally, theETEC cells were suspended in Dulbecco’s Modified Eagle’s Medium (DMEM) for theexperimental challenge with PIE cells. Live human ETEC O9, F4 pilus +, STp + kanamycin
resistant strain was used for in vivo experiments as described below.
2.2. Porcine Intestinal Epitheliocyte Cells
PIE cells are untransformed intestinal cultured cells. They were originally derived
from intestinal epithelium isolated from a non-suckling newborn pig [20]. PIE cells weremaintained in DMEM (Invitrogen Corporation, Carlsbad, CA, USA) supplemented with
10% fetal bovine serum, 100 mg/mL penicillin and 100 U/mL streptomycin at 37 ◦ C in a
5% CO2 atmosphere [21,36]. PIE cells were cultured in 250 mL flasks (1.0 × 106 cells) for
5 days changing the culture medium every 1–2 days. After reaching 80–90% confluence,cells were subcultured in 24-well flasks for immunostimulation assays as described below.
2.3. Immunomodulatory Assay in PIE Cells
Twelve-well type I collagen coated plates (Iwaki, Tokyo, Japan) were used to seed thePIE cells (3 × 104 cells/well) and they were cultured for 3 days. The medium was thenreplaced and the lactobacilli (108 cells/mL) were added. They were shaken in a microplatemixer and co-cultured for 48 h at 37 ◦ C in a 5% CO2 atmosphere. Each well was thenvigorously washed with medium at least 3 times to remove bacteria. The gene expressionof inflammatory cytokines (IL-6 and IL-8) and chemokines (CCL2, CCL8, CXCL5, CXCL9,CXCL10, and CXCL11), as well as negative regulators of TLR4 signaling (SIGIRR, Tollip,
A20, Bcl-3, IRAK-M, and MKP-1), were studied without any inflammatory challenge (basal
levels) or after a thermostable ETEC PAMPs challenge (5 × 107 cells/mL) for 12 h using
RT-PCR as described below.
2.4. Quantitative Expression Analysis by RT-PCR
We performed two-step real-time quantitative PCR to characterize the expression of
selected genes in PIE cells as described before [13,32]. The primers used in this study were
described previously [18,21,37]. The PCR cycling conditions were 2 min at 50 ◦ C, followedby 2 min at 95 ◦ C, and then 40 cycles of 15 s at 95 ◦ C, 30 s at 60 ◦ C, and 30 s at 72 ◦ C. The
reaction mixtures contained 5 µL of sample cDNA and 15 µL of master mix, including thesense and antisense primers. Expression of β-actin was used to normalize cDNA levels
for differences in total cDNA levels in the samples. In ETEC challenge experiments, a
relative index was calculated after normalization with β-actin and results were expressed
as normalized fold expression based on challenged control PIE cells set as 1.0.
2.5. ETEC Challenge in Mice
This study was carried out in strict accordance with the recommendations of the
Guide for the Care and Use of Laboratory Animals of the CERELA, Guide for Animal
Experimentation. Five-week-old female BALB/c mice were obtained from the closed colony
maintained at CERELA (Tucumán, Argentina). They were housed in plastic cages with
controlled room temperature (22 ± 2 ◦ C temperature, 55 ± 2% humidity) and mice were
fed ad libitum with a conventional balanced diet. Researchers and personnel specialized inanimal care and handling at CERELA ensured animal welfare. The health and behavior of
the animals were monitored twice a day. The tests for each parameter studied were carriedout in 5–6 mice per group. Animals were euthanized immediately after the time point wasreached using xylazine and ketamine. No signs of discomfort or pain were observed andthere were no deaths before the mice reached the end points.
L. plantarum CRL1506, CRL681 or TL2677 were administered orally to different groupsof mice for 5 consecutive days at a dose of 108 cells/mouse/day. On the sixth day, thelactobacilli-treated groups and the untreated control mice were orally inoculated with
200 mL of a bacterial suspension containing human ETEC O9, F4 pilus +, STp + kanamycin
resistant strain (1 × 109 cells) diluted with 0.1 M carbonate buffer (pH 9.0). Two days afterMicroorganisms 2023, 11, 63

5 of 17ETEC inoculation, the mice were sacrificed to collect jejunum, ileum, spleen, and liver
samples. The collected tissues were weighed and homogenized in BHI broth. Homogenates
were plated on MAC agar plates containing kanamycin for ETEC counts. The results wereexpressed as logarithm of colony forming units (CFU) per gram of organ.
2.6. Ex Vivo Peritoneal Macrophage Phagocytosis Assay
Peritoneal macrophages were collected aseptically from mice as previously described [18,38]. Briefly, the inner skin was exposed, and cold PBS supplemented with 10%
fetal calf serum by carefully injection into the peritoneal cavity. The liquid was collected,
and the macrophages were washed twice with PBS containing bovine serum albumin(BSA). Cells were adjusted to a concentration of 1 × 106 per ml. Phagocytosis was assessedusing heat-treated Saccharomyces boulardi. For this purpose, mixtures of opsonized yeast in
autologous mouse serum (10%) were added to 0.2 mL of macrophage suspension. Samples
were incubated for 30 min at 37 ◦ C. The percentage of phagocytosis was expressed as thepercentage of phagocytic macrophages in 200 cells counted using a light microscope.
2.7. Bactericidal Activity of Peritoneal Macrophages
The bactericidal activity (oxidative blast) of peritoneal macrophages was measured
using the nitro blue tetrazolium reduction test (NBT, Sigma-Aldrich, St. Louis, MO, USA)as previously described [18,38]. Briefly, peritoneal macrophages were obtained as describedabove and 200 µL of these cells were incubated with 120 µL of NBT reagent. Samples wereincubated first at 37 ◦ C for 10 min and then 10 min at room temperature. Then, NBT wasadded and incubated at 37 ◦ C for 20 min. In the presence of oxidative metabolites, NBT(yellow) is reduced to formazan, which forms a blue precipitate. Finally, the samples were
examined with a light microscope for blue precipitates. At random, 100 cells were counted
and the percentage of NBT positive (+) cells was determined.
2.8. Cytokine Concentrations
Concentrations of cytokines were determined in blood and intestinal samples fromlactobacilli-treated and control mice. Blood samples were obtained by cardiac puncture under anesthesia. Intestinal fluid samples were obtained as previously described (Indo et al.,2021). TNF-α, IL-6, IL-10, IFN-γ, chemokine KC (or CXCL1) and monocyte chemoattractant
protein 1 (MCP-1) concentrations were measured with enzyme-linked immunosorbent
assay (ELISA) kits following the manufacturer’s recommendations (R&D Systems, Minneapolis, MN, USA).
2.9. Statistical Analysis
Experiments were performed in triplicate and results expressed as the mean ± SD. For
the comparison of two groups, the Student’s t-test was used after the verification of normal
distribution. For the comparison of more than two groups, a one-way analysis of variance(ANOVA) was performed followed by and Tukey’s test. In all cases, a level of significanceof p < 0.05 was considered.
3. Results
3.1. Effect of L. plantarum Strains on the Expression of Cytokines and the Negative Regulators of
the TLR4 Signaling in PIE Cells
We evaluated whether L. plantarum CRL1506, CRL681 or TL2766 could modify thecytokine expression profile of PIE cells and whether the immunomodulatory property wasstrain specific. For this purpose, we comparatively analyzed the mRNA levels of IL-6 andthe chemokines IL-8, CCL2, CCL8, CXCL5, CXCL9, CXCL10, and CXCL11 in PIE cells
stimulated with these three bacterial strains (Figure 1). Stimulation of PIE cells with strains
CRL1506 or CRL681 increased the expression of IL-6, IL-8, CCL2, CCL8 and CXCL9, while
no significant differences were found in the levels of these inflammatory cytokines andchemokines between PIE cells treated with L. plantarum TL2677 and the control group.

Microorganisms 2023, 11, 63

We evaluated whether L. plantarum CRL1506, CRL681 or TL2766 could modify thecytokine expression profile of PIE cells and whether the immunomodulatory property
was strain specific. For this purpose, we comparatively analyzed the mRNA levels of IL6 and the chemokines IL-8, CCL2, CCL8, CXCL5, CXCL9, CXCL10, and CXCL11 in PIE
cells stimulated with these three bacterial strains (Figure 1). Stimulation of PIE cells with
6 of 17
strains CRL1506 or CRL681 increased the expression of IL-6, IL-8, CCL2, CCL8 and
CXCL9, while no significant differences were found in the levels of these inflammatory
cytokines and chemokines between PIE cells treated with L. plantarum TL2677 and thecontrol
the strain
CRL1506
was ableto increase
CXCL9
and compared
CXCL11 levels
Onlythegroup.
strain Only
CRL1506
was ableto increase
CXCL9
and CXCL11
levelsto thecomparedtothecontrolgroup,while
CXCL10levelsdecreasedsignificantly
in PIE
cells
control group, while CXCL10 levels decreased significantly in PIE cells treated
with
this
treated with this Lactiplantibacillus
There wasno significant
differencein chemokine
the levels
Lactiplantibacillus
strain. There wasstrain.
no significant
differencein the levelsof thisof this chemokine
among(Figure
the otheramong
the other groups1). groups (Figure 1).

FigureEffect of Lactiplantibacillus
Lactiplantibacillus plantarum
plantarumCRL681,
CRL681,CRL1506
CRL1506ororTL2677
TL2677ononthe
theexpression
expressionofof
Figure 1.
1. Effect
cytokines and chemokines
cytokines
chemokines inin porcine
porcine intestinal
intestinalepithelial
epithelial(PIE)(PIE)cells.
cells.PIE
PIEcells
cellswere
werestimulated
stimulatedwith
with
CRL681, CRL1506
CRL1506 or
CRL681,or TL2677
TL2677 strains
strains for
for48
48h.
h.Untreated
Untreatedcells
cellswere
wereusedusedasascontrols.
controls.The
Theexpression
expressionof cytokines
cytokines (IL-6)(IL-6) and
and
CCL8)
of
and chemokines
chemokines(CCL2,(CCL2,CXCL5,CXCL5,CXCL8,CXCL8,CXCL9,CXCL9,CXCL10,CXCL10,CXCL11,CXCL11,and
CCL8)were studied at 48 h after lactobacilli stimulation (basal). The results represent three independent
were studied at 48 h after lactobacilli stimulation (basal). The results represent three independent
experiments. Results are expressed as mean ± SD. Bold letters indicate significant differences whenexperiments. Results are expressed as mean ± SD. Bold letters indicate significant differences whencompared to the control group (p < 0.05).
compared to the control group (p < 0.05).

In addition, we evaluated the influence of the three L. plantarum strains on the exIn addition, we evaluated the influence of the three L. plantarum strains on the exprespression of the negative regulators of the TLR4 signaling in PIE cells (Figure 2). No signifsion of the negative regulators of the TLR4 signaling in PIE cells (Figure 2). No significant
icant differences were observed in the expression of SIGIRR and Tollip when untreateddifferences were observed in the expression of SIGIRR and Tollip when untreated control
control PIE cells and those treated with the different L. plantarum strains were compared.
PIE cells and those treated with the different L. plantarum strains were compared. In addiIn addition, L. plantarum CRL681 and CRL1506 were able to reduce the expression of A20
tion, L. plantarum CRL681 and CRL1506 were able to reduce the expression of A20 in PIE
in PIE cells and both strains increased the expression of Bcl-3 and IRAK-M. L. plantarum
cells and both strains increased the expression of Bcl-3 and IRAK-M. L. plantarum CRL1506
CRL1506 was the only strain capable of increasing the expression of MKP-1 compared to
was the only strain capable of increasing the expression of MKP-1 compared to the control
the control group (Figure 2).
group (Figure 2).

3.2. Effect of L. plantarum Strains on ETEC-Activated Innate Immune Response in PIE Cells
To study modulation of cytokines and chemokines in the context of inflammation,
PIE cells were treated with L. plantarum CRL681, CRL1506 or TL2677. Then, cells werechallenged with thermostable ETEC PAMPs. These molecular patterns can trigger TLR4
activation in this cell line, as we described previously [18,36]. Non-Lactiplantibacillus treated
PIE cells challenged with ETEC were used as controls. The ETEC PAMPs significantly
increased the expression of all inflammatory cytokines and chemokines in all experimentalgroups as shown in Figure 3, when compared to basal levels. The mRNA expression levelsof IL-6 were significantly higher in cells treated with CRL681 and CRL1506 strains. On
the other hand, the expressions of IL-8, CCL2, CXCL5 and CXCL9 were lower in PIE cells
treated with these strains compared to controls. Interestingly, only L. plantarum CRL1506
enhanced the expression of CCL8 (Figure 3). L. plantarum TL2677 increased the expressionof CXCL11 after exposure to ETEC, while strain CRL1506 reduced the expression of thisMicroorganisms 2023, 11, 63

7 of 17chemokine (Figure 3). No significant differences were observed in the levels of CXCL10
7 of 18
between the groups treated with lactobacilli and the control group after ETEC challenge(Figure 3).

Microorganisms 2023, 11, x FOR PEER REVIEW

FigureEffect ofof Lactiplantibacillus
Lactiplantibacillus plantarum
plantarumCRL681,
CRL681,CRL1506
CRL1506ororTL2677
TL2677ononthe
theexpression
expressionofof
Figure 2.
2. Effect
negative
the Toll-likeToll-like receptor
receptor (TLR)(TLR)signaling
signalingpathway
pathwayininporcine
porcineintestinal
intestinalepithelial
epithelial
negative regulators of the(PIE) cells.
cells. PIE cells were
(PIE)
were stimulated
stimulated with
withCRL681,
CRL681,CRL1506
CRL1506ororTL2677
TL2677strains
strainsforfor4848h.h.Untreated
Untreated
cells were
were used
used asas controls.
controls. The
cells
The expression
expressionof
ofnegative
negativeregulators
regulatorsofofthe
theTLR
TLRsignaling
signalingpathway
pathway
werestudied
at
48
h
after
lactobacilli
stimulation(basal).
The
results
represent
threewere studied at 48 h after lactobacilli stimulation (basal). The results represent threeindependent
independent
Microorganisms 2023, 11, x FOR PEERexperiments.
REVIEWof 18
Results are expressed as mean ± SD. Bold letters indicate significant differences8 whenexperiments. Results are expressed as mean ± SD. Bold letters indicate significant differences whencompared to the control group (p < 0.05).
compared to the control group (p < 0.05).

3.2. Effect of L. plantarum Strains on ETEC-Activated Innate Immune Response in PIE Cells
To study modulation of cytokines and chemokines in the context of inflammation,
PIE cells were treated with L. plantarum CRL681, CRL1506 or TL2677. Then, cells werechallenged with thermostable ETEC PAMPs. These molecular patterns can trigger TLR4
activation in this cell line, as we described previously [18,36]. Non-Lactiplantibacillus
treated PIE cells challenged with ETEC were used as controls. The ETEC PAMPs significantly increased the expression of all inflammatory cytokines and chemokines in all experimental groups as shown in Figure 3, when compared to basal levels. The mRNA expression levels of IL-6 were significantly higher in cells treated with CRL681 and CRL1506
strains. On the other hand, the expressions of IL-8, CCL2, CXCL5 and CXCL9 were lower
in PIE cells treated with these strains compared to controls. Interestingly, only L. plantarum
CRL1506 enhanced the expression of CCL8 (Figure 3). L. plantarum TL2677 increased theexpression of CXCL11 after exposure to ETEC, while strain CRL1506 reduced the expression of this chemokine (Figure 3). No significant differences were observed in the levelsof CXCL10 between the groups treated with lactobacilli and the control group after ETEC
challenge (Figure 3).

FigureEffectof
ofLactiplantibacillus
Lactiplantibacillusplantarum
plantarumCRL681,
CRL681,CRL1506
CRL1506ororTL2677
TL2677ononthe
theexpression
expressionofof
Figure 3. Effectcytokines
coli
cytokinesandandchemokines
chemokinesin
inporcine
porcineintestinal
intestinalepithelial
epithelial(PIE)(PIE)cells
cellsafter
afterEnterotoxigenic
EnterotoxigenicEscherichia
Escherichia
coli (ETEC)
challenge.
PIE werecells werepre-treatedwith CRL681,
CRL1506
or TL2677strainsh(ETEC)
challenge.
PIE cells
pre-treatedwith CRL681,
CRL1506
or TL2677strains
for 48 hfor
and48thenand then stimulatedwith heat-stable
ETEC pathogen-associatedpatterns (PAMPs).
Nonstimulatedwith heat-stable
ETEC pathogen-associatedmolecularmolecularpatterns (PAMPs).
Non-lactobacilli
lactobacilli treated cells were used as controls. The expression of cytokines (IL-6) and chemokines
treated cells were used as controls. The expression of cytokines (IL-6) and chemokines (CCL2, CXCL5,(CCL2, CXCL5, CXCL8, CXCL9, CXCL10, CXCL11, and CCL8) were studied at 12 h after heat-stableCXCL8, CXCL9, CXCL10, CXCL11, and CCL8) were studied at 12 h after heat-stable ETEC PAMPsETEC PAMPs challenge. The results represent three independent experiments. Results are exchallenge.
results
threeindicate
independent
experiments.
Results
arecompared
expressedtoasthe
mean
± SD.
pressed asThemean± SD.represent
Bold letterssignificant
differences
whencontrol
Bold
letters
indicatesignificant
differenceswhencomparedtothecontrolgroup(p<0.05).
group (p < 0.05).

When the negative regulators of the TLR signaling pathway were investigated after
exposure to ETEC PAMPs, it was found that only L. plantarum CRL1506 reduced the expressions of A20 and Bcl-3 (Figure 4). This strain also increased the levels of MKP-1 in
ETEC-challenged PIE cells. No significant differences were observed in the levels of theMicroorganisms 2023, 11, 63

coli (ETEC) challenge. PIE cells were pre-treated with CRL681, CRL1506 or TL2677strains for 48 hand then stimulated with heat-stable ETEC pathogen-associated molecular patterns (PAMPs). Nonlactobacilli treated cells were used as controls. The expression of cytokines (IL-6) and chemokines(CCL2, CXCL5, CXCL8, CXCL9, CXCL10, CXCL11, and CCL8) were studied at 12 h after heat-stable
ETEC PAMPs challenge. The results represent three independent experiments. Results are ex8 of 17pressed as mean ± SD. Bold letters indicate significant differences when compared to the control
group (p < 0.05).

When thethe negative
negative regulators
regulators ofof thethe TLRTLR signaling
signaling pathway
pathway were
were investigatedinvestigated after
When
after
exposureto
ETEC
PAMPs,
it
was
found
that
only
L.
plantarum
CRL1506
reducedthe theexexposure to ETEC PAMPs, it was found that only L. plantarum CRL1506
reducedpressions
of
A20
and
Bcl-3
(Figure4).
Thisstrainalso
increased
thelevelsof
MKP-1
inexpressions of A20 and Bcl-3 (Figure 4). This strain also increased the levels of MKP-1 in
ETEC-challenged PIE
theETEC-challenged
PIE cells.
cells. No
No significant
significant differences
differences were
were observed
observed inin thethe levels
levels ofof theother mediators
mediators analyzed
analyzed inin thisthis study
study (Figure(Figure 4).
4).
other

Figureplantarum
CRL681,
CRL1506
or TL2677
on the expressionof negative
Figure 4.
4. Effect
EffectofofLactiplantibacillus
Lactiplantibacillus
plantarum
CRL681,
CRL1506
or TL2677
on the expressionofregulatorsof the Toll-likesignaling
porcinein
intestinal
cells
negative regulatorsof thereceptor
Toll-like(TLR)
receptor(TLR) pathway
signalingin
pathway
porcine epithelial
intestinal(PIE)epithelial(PIE)Enterotoxigenic
cells after Enterotoxigeniccoli (ETEC)
challenge.
PIE cells werepre-treated
with
after
Escherichia coliEscherichia(ETEC) challenge.
PIE cells
were pre-treatedwith CRL681,
CRL1506
CRL681,
or48
TL2677for 48 h andstimulatedwithheat-stable ETEC pathogenor
TL2677CRL1506
strains forh andstrainsthen stimulated
withthenheat-stable
ETEC
pathogen-associatedmolecular
patterns (PAMPs). Non-lactobacilli treated cells were used as controls. The expression of negative
regulators of the TLR signaling pathway were studied at 12 h after heat-stable ETEC PAMPs challenge.
The results represent three independent experiments. Results are expressed as mean ± SD. Bold letters
indicate significant differences when compared to the control group (p < 0.05).

3.3. L. plantarum CRL681 and CRL1506 Modulate Intestinal Immunity In Vivo
In addition, the ability of the different L. plantarum strains to stimulate macrophages
was evaluated. For this purpose, ex vivo analysis of the phagocytic and bactericidal activityof the peritoneal macrophages were carried out. L. plantarum strains CRL681 and CRL1506
significantly increased the phagocytic activity of peritoneal macrophages, while this effect
was absent in the strain TL2677 (Figure 5). In addition, a significant difference in thepercentage of phagocytosis between the strains CRL681 and CRL1506 was observed, thelatter strain showing higher phagocytic activity (Figure 5). To study the activation of
the respiratory burst in peritoneal macrophages, we used the NBT method as previously
described [18]. The treatment with the CRL1506 strain was more effective to enhance thepercentage of NBT+ cells in the macrophage population obtained from the peritoneal cavity
than the treatment with the CRL681 strain (Figure 5). Of note, L. plantarum TL2677 did not
induce significant changes compared to the control group (Figure 5).
We also analyzed the cytokine concentrations in the intestinal fluid and serum obtained
from mice treated with lactobacilli to determine the local and systemic effects inducedby the L. plantarum strains (Figure 6). Both L. plantarum CRL681 and CRL1506 increased
the levels of intestinal and serum IFN-γ. The concentrations of this cytokine found in
the group stimulated with CRL1506 was higher than the CRL681 group (Figure 6). In
addition, L. plantarum CRL681 and CRL1506 increased the level of intestinal TNF-α, while
no differences were observed for serum TNF-α between the groups. Increased levels of
IL-10 were found in both the intestinal fluid and serum of CRL1506- and CRL681-treatedmice. However, serum levels of this immunoregulatory cytokine were significantly higher
in mice treated with L. plantarum CRL1506 compared to those that received the CRL681
strain (Figure 6). No significant differences were observed between mice treated with

Microorganisms 2023, 11, 63

In addition, the ability of the different L. plantarum strains to stimulate macrophages
was evaluated. For this purpose, ex vivo analysis of the phagocytic and bactericidal activity of the peritoneal macrophages were carried out. L. plantarum strains CRL681 and
CRL1506 significantly increased the phagocytic activity of peritoneal macrophages, while
this effect was absent in the strain TL2677 (Figure 5). In addition, a significant differencein the percentage of phagocytosis between the strains CRL681 and CRL1506 was ob9 of 17
served, the latter strain showing higher phagocytic activity (Figure 5). To study the activation of the respiratory burst in peritoneal macrophages, we used the NBT method as
previously described [18]. The treatment with the CRL1506 strain was more effective toenhance the percentage of NBT+ cells in the macrophage population obtained from theL. plantarum
controlwhenconcentrationsof intestinal and serum
peritoneal
cavityTL2677
than theandtreatment
withanimals
the CRL681
strainthe(Figure5). Of note, L. plantarum
TL2677 didnotanalyzed
induce significant
changescytokines
were(Figure6). compared to the control group (Figure 5).

Figureof Lactiplantibacillus
plantarum CRL681,
CRL1506CRL681,
or TL2677 on
peritonealor
macroFigure5. Effect
5. Effectof Lactiplantibacillus
plantarum
CRL1506
TL2677 on peritoneal
phages activities. Mice were orally treated with L. plantarum CRL681, CRL1506 or TL2677 (108macrophages activities. Mice were orally treated with L. plantarum CRL681, CRL1506 or TL2677 (108cells/mouse per day for 5 consecutive days). Untreated mice were used as controls. One day after
cells/mouseper
day for 5 consecutivedays).
Untreatedmiceburst)
wereactivities
used asofcontrols.
One day after thethelast lactobacilli
administration,
phagocytic andbactericidal(oxidative
peritoneal
were determined. The
results represent
three independent
experiments.
Results
last macrophages
lactobacilli administration,
phagocytic
and bactericidal(oxidative
burst)
activities of peritoneal
are expressed as mean ± SD. Bold letters indicate significant differences when compared to the conmacrophages
were determined. The results represent three independent experiments. Results aretrolgroup (p < 0.05).

as mean ± SD. Bold letters indicate significant differences whento the control
Microorganisms 2023, 11, x FOR PEERexpressed
REVIEW
10 comparedof 18
We(palso
analyzed the cytokine concentrations in the intestinal fluid and serum obgroup< 0.05).
tained from mice treated with lactobacilli to determine the local and systemic effects induced by the L. plantarum strains (Figure 6). Both L. plantarum CRL681 and CRL1506 increased the levels of intestinal and serum IFN-γ. The concentrations of this cytokine foundin the group stimulated with CRL1506 was higher than the CRL681 group (Figure 6). In
addition, L. plantarum CRL681 and CRL1506 increased the level of intestinal TNF-α, while
no differences were observed for serum TNF-α between the groups. Increased levels of
IL-10 were found in both the intestinal fluid and serum of CRL1506- and CRL681-treatedmice. However, serum levels of this immunoregulatory cytokine were significantly higher
in mice treated with L. plantarum CRL1506 compared to those that received the CRL681
strain (Figure 6). No significant differences were observed between mice treated with L.
plantarum TL2677 and control animals when the concentrations of intestinal and serum
cytokines were analyzed (Figure 6).

Figure 6.6.
Effectof Lactiplantibacillus
plantarumplantarum
CRL681, CRL1506
or TL2677
on intestinal
and serum
FigureEffectof Lactiplantibacillus
CRL681,
CRL1506
or TL2677
on intestinal and serum
cytokines of adult immunocompetent mice after enterotoxigenic Escherichia coli (ETEC) challenge.
cytokinesof adult immunocompetent mice after enterotoxigenic
Escherichia
coli (ETEC) challenge.
Mice were orally treated with L. plantarum CRL681, CRL1506 or TL2677 (108 cells/mouse per day for89Micewere orally
treatedwith L. orally
plantarum
CRL681,
CRL1506
TL2677
cells/mouse per day
5 consecutivedays) andthen challenged
with ETEC
F4 strain(10 cells). orMicewith no (10lactoba9 cells). Mice with nocilli treatment
and challenged
with ETEC
used as controls.
afterF4
thestrain
challenge,for
5 consecutivedays) andthen werechallenged
orally Two
withdays
ETEC(10theconcentrations of tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and interleukin (IL)-10 in inteslactobacilli
treatment
andchallenged
withTheETEC
wereused three
as controls.
Twoexperidays after the challenge,
tinal fluid andserum were
determinedby ELISA.
results
represent
independent
ments.
Results are expressed
as mean± SD. Bold
letters(TNF)-α,indicate significant
differenceswhen
comtheconcentrationsof tumor
necrosisfactorinterferon(IFN)-γ,andinterleukin (IL)-10 inpared to the control group (p < 0.05).
intestinal
fluid and serum were determined by ELISA. The results represent three independent
experiments.
as mean± SD.ofBold
indicate
3.4. L. plantarumResults
CRL681are
andexpressed
CRL1506 prevent
the Spread
ETEClettersand Modulatethesignificant differences whencomparedthe control
group(pBacterial< 0.05).Challenge
Expression ofto
Intestinal
Cytokines
after

In order to evaluate the effect of L. plantarum strains on resistance to ETEC infection,

3.4.
plantarum
CRL681
CRL1506
preventileum,
the Spreadof ETEC
and
Modulate the Expression
bodyL.
weightloss andbacterialandcountsin the jejunum,
liver andspleen ofinfectedofIntestinal
Cytokines
after
mice
were determined
twodaysBacterial
after the Challenge
challenge. We evaluated body weight loss tostudy In
theordergeneraltohealthstate ofmice
(Figure7).plantarum
The infection
with ETEC
significantlyto ETEC infection,
evaluatetheeffectof L.
strains
on resistanceincreased the body weight loss of mice we described previously [37]. Of note, mice treatedbodyweight loss and bacterial counts in the jejunum, ileum, liver and spleen of infectedwith the CRL1506 and CRL681 strains had significantly lower percentages of body weight
mice
were
determinedtwo
daysandafter
the challenge.
Weto evaluatedweight loss toloss thancontrols.
L. plantarum
CRL681
CRL1506
were also able
significantly body
restudy
thecounts
generalhealthandstateof mice(Figure7). Theinfectionwith ETEC significantly
duce ETEC
in jejunum
ileum
compared
to controls(Figure7). Furthermore,both lactobacilli
prevented
themicespreadweof thepathogenpreviously
to the spleen [37].
and liver.
increased
the treatments
body weightloss ofdescribed
Of note, mice treated
There thewereCRL1506
no significant
body weightloss and the pathogen’s
counts ob- of body weightwithanddifferences
CRL681instrains
had significantly
lower percentages
served in the different organs of the mice treated with TL2677 and the control group (Figloss
than controls. L. plantarum CRL681 and CRL1506 were also able to significantly reduceure 7).
ETEC counts in jejunum and ileum compared to controls (Figure 7). Furthermore, bothlactobacilli treatments prevented the spread of the pathogen to the spleen and liver. There
were no significant differences in body weight loss and the pathogen’s counts observed in
the different organs of the mice treated with TL2677 and the control group (Figure 7).

Microorganisms 2023, 11, 63

increased the body weight loss of mice we described previously [37]. Of note, mice treatedwith the CRL1506 and CRL681 strains had significantly lower percentages of body weightloss than controls. L. plantarum CRL681 and CRL1506 were also able to significantly reduce ETEC counts in jejunum and ileum compared to controls (Figure 7). Furthermore,both lactobacilli treatments prevented the spread of the pathogen to the spleen and liver.
There were no significant differences in body weight loss and the pathogen’s counts observed in the different organs of the mice treated with TL2677 and the control group (Figure 7).

10 of 17

Figure 7. Immunomodulatory effect of Lactiplantibacillus plantarum CRL681, CRL1506 or TL2677 in
11 of 18
mice in response to enterotoxigenic Escherichia coli (ETEC) challenge. Mice were orally treated with
8
L. plantarum CRL681, CRL1506 or TL2677 (10 cells/mouse per day for 5 consecutive days) and thenchallenged
orally with ETEC F4 strain (109 cells). Mice with no lactobacilli treatment and challenged
Figure 7. Immunomodulatory effect of Lactiplantibacillus plantarum CRL681, CRL1506 or TL2677 in
with
ETEC
were
used as controls.
ETECcolicountsjejunum,Miceileum,
spleenmice in
response
to enterotoxigenic
Escherichia(ETEC)inchallenge.
wereliver,orally andtreatedwith were determined
L. plantarum
CRL681,or TL2677(108 arecells/mouseday
5 consecutivedays) andsignificant
thentwodays after
theCRL1506
challenge.
Valuesmeansper±SD.forBold
letters indicate
differences
challenged orally with ETEC F4 strain (109 cells). Mice with no lactobacilli treatment and challenged
when
compared
to
the
ETEC
controlgroup(p<0.05).
with ETEC were used as controls. ETEC counts in jejunum, ileum, liver, and spleen were determined

Microorganisms 2023, 11, x FOR PEER REVIEW

two days after the challenge. Values are means ± SD. Bold letters indicate significant differences
when comparedto thecontrolIL-6,
groupIFN-γ,(p < 0.05).MCP-1, KC and IL-10 were also quantified in the
The levels
ofETECTNF-α,

gut mucosa of mice treated with lactobacilli and challenged with ETEC. Both L. plantarum
The levels of TNF-α, IL-6, IFN-γ, MCP-1, KC and IL-10 were also quantified in the
CRL681 and CRL1506 significantly reduced the intestinal levels of TNF-α, KC and MCP-1
gut mucosa of mice treated with lactobacilli and challenged with ETEC. Both L. plantarumincomparison
withthe controls,
being
the CRL1506
effective to induce
CRL681and CRL1506
significantly
reducedthe intestinallevels ofstrain
TNF-α,theKC mostand MCPthe
decreaseof
thesecytokines(Figure8).
Micetreatedwithstrains
CRL681or CRL1506
1 in comparison with the controls, being the CRL1506 strain the most effective to induce
the decreaseof thesecytokinesIL-10(Figure8). Mice treatedthanwith strains
CRL681or only
CRL1506
showedhigherintestinal
concentrations
controls,
while
CRL1506 increased
showed levels
higher compared
intestinal IL-10
concentrations
than controls,
onlysignificant
CRL1506 in-differences wereIFN-γ
to thecontrol group(Figurewhile
8). Nocreased IFN-γ levels compared to the control group (Figure 8). No significant differences
observed in IL-6 levels between the studied groups. In addition, no differences were
were observed in IL-6 levels between the studied groups. In addition, no differences weredetected
intestinal
cytokines
levels between
group
with
L. plantarum TL2677
detected inin
intestinal
cytokines
levels between
the group thetreatedwith treated
L. plantarum
TL2677andthecontrol
controlgroup(Figure8).
and thegroup(Figure8).

Figure 8.8.Immunomodulatory
effect of
Lactiplantibacillus
plantarum CRL681,
CRL1506
or TL2677
inFigureImmunomodulatory
effectof Lactiplantibacillus
plantarum
CRL681,
CRL1506
or TL2677 inmice in response to enterotoxigenic Escherichia coli (ETEC) challenge. Mice were orally treated withmice in response to enterotoxigenic Escherichia
coli (ETEC) challenge. Mice were orally treated with
8
L. plantarum CRL681, CRL1506 or TL2677 (10 cells/mouse per day for 5 consecutive days) and then9 cells). MiceL.
plantarum
CRL681,
CRL1506(108withcells/mouseper
day forand
5 consecutivedays) and thenchallenged
orallywith ETEC
F4 strainor(10TL2677
no lactobacilli
treatmentchallenged
9 cells).
with ETEC wereusedwith
as controls.
levelsof tumor
necrosisfactor(TNF)-α, interferon
challenged
orallyETECTheF4 intestinal
strain (10Micewith nolactobacilli
treatment and challenged(IFN)-γ, interleukin (IL)-6, IL-10, chemokine KC (or CXCL1), and monocyte chemoattractant protein
with
ETEC were used as controls. The intestinal levels of tumor necrosis factor (TNF)-α, interferon
1 (MCP-1) were determined two days after the challenge with ETEC. Values are means ± SD. Bold
(IFN)-γ,interleukin(IL)-6,
IL-10,when
chemokine(orETECCXCL1),
monocyte
letters indicate
significant
differencescomparedKC
to thecontrolandgroup(p < 0.05). chemoattractant protein
1 (MCP-1) were determined two days after the challenge with ETEC. Values are means ± SD. Bold
4. Discussion
letters indicate significant differences when compared to the ETEC control group (p < 0.05).

LAB of various species can be used as probiotics for animals with the aim to control
pathogenic microorganisms and improve natural defense mechanisms, reducing healthproblems and therefore increasing productivity [39,40]. L. plantarum is among the mostversatile species used for decades in the food industry either as starter cultures or probiotics [25]. They are applied as starter cultures to produce cheeses, sausages, olives and awide variety of fermented foods and beverages, contributing to their organoleptic properties, flavor and texture. This outstanding versatility and metabolic activity can be ex-Microorganisms 2023, 11, 63

11 of 17

4. Discussion
LAB of various species can be used as probiotics for animals with the aim to control
pathogenic microorganisms and improve natural defense mechanisms, reducing healthproblems and therefore increasing productivity [39,40]. L. plantarum is among the mostversatile species used for decades in the food industry either as starter cultures or probiotics [25]. They are applied as starter cultures to produce cheeses, sausages, olives and awide variety of fermented foods and beverages, contributing to their organoleptic properties, flavor and texture. This outstanding versatility and metabolic activity can be explainedby its big genome (2.91 to 3.7 Mbp in length) compared to other LAB such as Latilactobacillus
curvatus or Lactobacillus sakei which account for less than 1.9 Mbp [41,42] This feature contributes to its survival capability in a wide range of environmental niches including plants,
fermented foods, and the gastrointestinal tract humans and other mammals [22,23]. The
increasing significance as probiotic of this species are mainly linked to health promotion inhumans and animals [24,43–49]. However, their beneficial effects are strain dependent and
not universal. Therefore, the probiotic properties need to be characterized on a strain level.
In this context, the immunobiotic potential of two L. plantarum strains with different
origins and probiotic/technological properties was studied and compared to evaluate theircapability to improve the resistance to ETEC infection. L. plantarum CRL681, originallyisolated from fermented sausages, has an efficient acidogenic activity that guarantees safety
and texture development during ripening [31,44]. In addition, detailed peptidomic studies
confirmed its peptidogenic ability and its capacity to increase free amino acid contents
in meat or fermented-meat models [31–33]. The CRL681 strain is capable of degrading
biogenic amines in vitro and lacks the ability to produce them [40]. Moreover, this strain
has remarkable bioprotective potential due to the high inhibitory activity toward E. coli
O157:H7 [35]. On the other hand, L. plantarum CRL1506, originally isolated from goat milk,
has demonstrated to possess remarkable immunomodulatory activities in the context ofantiviral immunity [26–29,46], although its capacity to modulate antibacterial immune
responses in the intestinal tract has not been explored in depth.
In the present study, we demonstrated that both CRL681 and CRL1506 strains canmodulate the intestinal immune response in steady-state conditions. L. plantarum CRL681
and CRL1506 triggered the expression of IL-6 and IL-8 in porcine IECs. Furthermore, miceorally treated with these strains had significantly improved levels of IFN-γ and TNF-α in
the intestine as well as enhanced activity of peritoneal macrophages. Studies evaluating theeffect of lactobacilli strains with the capacity to reduce the severity of intestinal infections
found that the most remarkable effect was the increase in the intestinal levels of TNF-α,
IFN-γ, IL-1β, IL-6, and IL-12 for the mice treated with the probiotic strains, as well as thephagocytic activity of intestinal and peritoneal macrophages [47,48]. In vitro and ex vivo
studies in a primary culture of IECs demonstrated that probiotic lactobacilli interact withthese cells and induce release of IL-6 [47,49]. This cytokine was shown to regulate thesurvival and proliferation of IECs as well as to stimulate immune cells in the intestinal
mucosa [50]. Autophagy in IECs is involved in the homeostatic control of cell deathand differentiation. It was shown that IECs have a high basal level of autophagy that isregulated by TLR-mediated IL-8 production [51]. On the other hand, probiotic bacteria
can stimulate macrophages by increasing their phagocytic activities and their capacity to
produce cytokines like IFN-γ and TNF-α. This macrophage activity is essential for theprotection against infections [52]. Thus, our results suggest that both CRL681 and CRL1506
strains have the capacity to interact with IECs and macrophages in the gut, reinforcing theepithelial defenses and stimulating immune responses. In fact, the ability of L. plantarum
CRL681 and CRL1506 to beneficially modulate intestinal immunity was put into greater
evidence in the experiments in which challenges with ETEC were performed.
In this work, using the in vitro PIE cell system we observed that L. plantarum CRL681
and CRL1506 differentially modulated the innate immune responses of porcine IECs
triggered by ETEC challenge. The CRL1506 and CRL681 strains modulated the expressionof inflammatory cytokines (IL-6) and chemokines (IL-8, CCL2, CXCL5 and CXCL9) inMicroorganisms 2023, 11, 63

12 of 17

ETEC-stimulated PIE cells. Furthermore, our studies in the mice model of ETEC infection
demonstrated in vivo the ability of L. plantarum CRL681 and CRL1506 to beneficiallyregulate the immune response against this pathogen. In our hands, the oral treatmentof mice with CRL681 or CRL1506 strains significantly reduced body weight loss as wellas ETEC counts in jejunum and ileum and prevented the spread of the pathogen to thespleen and liver. This protective effect could be related to a differential regulation ofcytokines and chemokines in the intestinal mucosa, particularly in IECs. Our results are inline with previous works demonstrating that probiotic microorganisms can regulate theimmune response against ETEC in both mice models [13,14] and pigs [15–17] as well as
in IECs [53,54]. Lacticaseibacillus rhamnosus GG and Bifidobacterium animalis MB5 protected
human Caco-2 cells from the ETEC K88-associated inflammation by reducing IL-1β and
TNF-α and enhancing TGF-β1 expression [53,55]. Enterococcus faecium HDRsEf1 has beenshown to have positive effects on piglet diarrhea, and studies performed in the porcine
IPEC-J2 cell line demonstrated that this effect is partially related to its ability to reduce
the expression of IL-8 after ETEC challenge, protecting IECs from the acute inflammatory
response [54]. In addition, it was shown that the porcine IECs line IPEC-1 treated with
L. plantarum CGMCC 1258 had reduced expressions of IL-1α, IL-6, IL-8 and TNF-α after thechallenge with ETEC K88. This effect was associated with the ability of the CGMCC 1258
strain to regulate MAPK and NF-κB signaling pathways [56]. Our previous studies in PIE
cells showed a reduction in the activation of NF-κB and MAPK signaling pathways and in
the expression of some inflammatory cytokines and chemokines in ETEC-challenged PIE
cells, preventively stimulated with Lactobacillus jensenii TL2937 [21], or Bifidobacterium breve
M-16V [36].
Then, our results suggest that the improved intestinal epithelial defenses and innate
immunity induced by L. plantarum CRL681 and CRL1506 would increase the clearanceof ETEC and at the same time, protect the host against detrimental inflammation. The
activation of TLR4 in the intestinal mucosa induce the production of cytokines to stimulate
the recruitment and activation of inflammatory cells. Although this mechanism is a key
primary line of host defense, prolonged or dysregulated proinflammatory response maylead to tissue damage and dysfunction [57]. Thus, the reduction of the intestinal levels of
TNF-α, MCP-1 and KC induced by the CRL681 and CRL1506 strains could indicate a better
control of inflammation and its detrimental effects. Furthermore, it was observed thatmice treated with L. plantarum CRL681 or CRL1506 had improved levels of intestinal IL-10
both at steady state and after the challenge with ETEC. Studies performed in healthy adultvolunteers challenged with ETEC demonstrated that higher pre-challenge concentrationsof IL-10 were associated with protection from ETEC diarrhea [9]. In addition, in agreementwith our results it was reported that the treatment of mice with L. plantarum CCFM1143
was able to alleviate diarrhea caused by ETEC infection, and that this beneficial effect was
associated with reductions of TNF-α and improvements of IL-10 [58]. Furthermore, in linewith the relevant role of IL-10 in controlling TNF-α production, our studies showed that inbaseline determinations the levels of this regulatory cytokine increased only 1.4 times inanimals treated with CRL1506 or CRL681 strains compared to controls and was not able
to decrease the levels of the inflammatory cytokine. In contrast, in the ETEC-challenge
experiments, IL-10 was augmented more than 10-fold compared to baseline values and1.6-fold between CRL1506 and CRL681 versus controls.
We have previously demonstrated that the beneficial effects of immunobiotic bacteria
in the context of TLR4-triggered inflammation are mediated by a differential modulation ofnegative regulators of the TLR signaling pathway [21,36]. Then, we also evaluated here theability of L. plantarum CRL681 or CRL1506 to modulate the expression of SIGIRR, Tollip,
A20, Bcl-3, IRAK-M, and MKP-1 in porcine IECs. We found that both strains upregulated theexpression of IRAK-M and reduced A20 in PIE cells without ETEC challenge. In addition,
CRL1506 and CRL681 increased the expressions of MKP-1 and Bcl-3, respectively. The TLR
negative regulators play important roles in the maintenance of intestinal hemostasis as wellas in the control of immune responses against pathogens. IRAK-M exerts its regulatory

Microorganisms 2023, 11, 63

13 of 17

effect by acting on the TRAF6/IRAK-1 complex, diminishing the activation of NF-κB
and MAPK pathways [59]. It was described that LPS challenge induce the expression of
IRAK-M, and that the tolerance to TLR4 activation is diminished in IRAK-M-deficient
cells [60]. On the other hand, it was reported that A20 [61,62] and Bcl-3 [63] regulate TLR4
signaling pathway by inducing the inhibition of NF-κB activation while MKP-1 inactivates
the MAPK p38 signaling pathway [64]. Thus, it is tempting to speculate that lactobacilli
would induce a differential expression of negative regulators of the TLR4 pathway inthe intestinal epithelium, and when the challenged with ETEC is induced, this signaling
pathway would be differentially regulated allowing an efficient induction of an antibacterial
state in the gut, and the protection against the inflammatory-mediated damage.
Of note, the immunostimulatory activity of the CRL1506 and CRL681 strains were
not achieved by L. plantarum TL2677 in porcine IECs. In addition, the protective effectof L. plantarum CRL681 and CRL1506 against ETEC infection was not observed for the
TL2677 strain in mice, highlighting that the immunomodulatory effects are strain specific.
Furthermore, the analysis of the immunomodulatory activities in steady state conditions
as well as the immune responses triggered by ETEC challenge showed that L. plantarum
CRL1506 is more efficient than CRL681 strain to modulate mucosal immunity. In line with
our results, it was shown that different strains had individual capacities to regulate TLR4NF-κB signaling pathway and the expression of IL-8 in HEK cells as well as to regulatetrans-epithelial electrical resistance and tight junction integrity in LPS-challenged Caco-2monolayers [65]. The different abilities of the L. plantarum strains studied here to influence
the immune response against ETEC could be associated with their distinct capacity tomodulate negative regulators of TLR. While the TL2677 did not modulate the expressionof TLR negative regulators, L. plantarum CRL1506 was more efficient than the CRL681
strain to upregulate IRAK-M expression and diminish A20. Moreover, when the TLR
negative regulators were evaluated in PIE cells challenged with ETEC, only cells treatedwith L. plantarum CRL1506 showed differences in the expressions of A20, Bcl-3 and MKP-1.
Interestingly, despite the more notable changes in the expression of immune factors bothin vitro and in vivo induced by the CRL1506 strain, no significant differences in ETEC
counts were observed when compared to mice treated with L. plantarum CRL681. This may
be due to the fact that in this work only one post-infection point was studied. Perhaps astudy of the kinetics of ETEC clearance could find differences between the two treatments.
In addition, a further study to evaluate whether a combination of both L. plantarum CRL1506
and CRL681 could improve immunity more effectively than individual strains and induce
a more efficient clearance of ETEC in mice intestine would be of value. Another important
point for future studies is to find the bacterial molecules and immune receptors involvedin the induction of TLR negative regulators in the intestinal epithelium by L. plantarum,
which also explain the differences between the strains. Our recent comparative genomic
study showed a great variability in the predicted surface proteins of L. plantarum strains,
including CRL1506 and CRL681 [30]. These results suggest that the surface molecules could
be involved in their differential ability to modulate the intestinal innate immune response
against ETEC.
5. Conclusions
Our results demonstrated that the improved intestinal epithelial defenses and innate
immunity induced by L. plantarum CRL1506 and CRL681 would increase the clearance of
ETEC and at the same time, the differential expression of negative regulators of the TLR4
pathway in the intestinal epithelium allowing the establishment of an antibacterial state in
the gut, and the protection against the inflammatory-mediated damage. These findings
constitute valuable features for a future probiotic culture for animal feed able to improve
the resistance to ETEC infection. The preliminary studies carried out in this work using
porcine IECs and mice provide the scientific basis for carrying out in vivo studies in pigs toreliably demonstrate the protective potential of CRL1506 and CRL681 strains against ETEC
infection in the porcine host.

Microorganisms 2023, 11, 63

14 of 17

Author Contributions: A.B. carried out the experiments, analyzed the results, contributed to thediscussion, and wrote the paper. J.V. conceived the idea of the project, carried out the experiments,
analyzed the results, contributed to the discussion, wrote the paper, and participated in funding
acquisition. L.A. carried out the experiments, analysed the results and contributed to the discussion.
M.T. contributed to the discussion and wrote the paper. M.E. carried out the experiments, analyzed
the results, analysed the results and contributed to the discussion. K.F. carried out the experiments,
analyzed the results, contributed to the discussion and wrote the paper. S.Q.-V. contributed to thediscussion and wrote the paper. S.F. coordinate, analyzed and discussed the results, wrote the paper,and participated in funding acquisition. H.K. coordinate, analyzed and discussed the results, wrote
the paper, and participated in funding acquisition. All authors have read and agreed to the publishedversion of the manuscript.
Funding: This study was supported by the research program on development of innovative technology grants (JPJ007097) from the Project of the Bio-oriented Technology Research Advancement
Institution (BRAIN), and by a Grant-in-Aid for Scientific Research (A) (19H00965) from the Japan
Society for the Promotion of Science (JSPS), and by Japan Racing Association (JRA) Livestock Industry Promotion Project, and by the Association for Research on Lactic Acid Bacteria to HK. This
study was also supported by ANPCyT–FONCyT Grant PICT-2016-0410 to JV, Grant PICT-2018-02249;
PICT-2018-0664 to SF and by JSPS Core-to-Core Program, A. Advanced Research Networks entitledEstablishment of international agricultural immunology research-core for a quantum improvement
in food safety, and by AMED Grant Number JP21zf0127001. Mikado Tomokiyo was supported by JST
SPRING, Grant Number JPMJSP2114. Kohtaro Fukuyama was supported by JST, the establishmentof university fellowships towards the creation of science technology innovation, Grant Number
JPMJFS2102.
Institutional Review Board Statement: Experiments with mice were performed in accordance withthe guide for the care and use of laboratory animals and were approved by the CERELA-CONICET
Animal Care and Ethics Committee under the BIOT-CRL/19 protocol.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are contained within this article.
Conflicts of Interest: The authors declare that the research was conducted in the absence of any
commercial or financial relationships that could be construed as a potential conflict of interest.

References
1.

2.

3.

4.
5.

6.
7.
8.

9.

Khalil, I.A.; Troeger, C.; Blacker, B.F.; Rao, P.C.; Brown, A.; Atherly, D.E.; Brewer, T.G.; Engmann, C.M.; Houpt, E.R.; Kang, G.;
et al. Morbidity and Mortality Due to Shigella and Enterotoxigenic Escherichia coli Diarrhoea: The Global Burden of Disease Study
1990–2016. Lancet Infect. Dis. 2018, 18, 1229–1240. [CrossRef] [PubMed]
Laird, T.J.; Abraham, S.; Jordan, D.; Pluske, J.R.; Hampson, D.J.; Trott, D.J.; O’Dea, M. Porcine Enterotoxigenic Escherichia coli:
Antimicrobial Resistance and Development of Microbial-Based Alternative Control Strategies. Vet. Microbiol. 2021, 258, 109117.
[CrossRef] [PubMed]
Wu, Q.; Cui, D.; Chao, X.; Chen, P.; Liu, J.; Wang, Y.; Su, T.; Li, M.; Xu, R.; Zhu, Y.; et al. Transcriptome Analysis Identifies
Strategies Targeting Immune Response-Related Pathways to Control Enterotoxigenic Escherichia coli Infection in Porcine Intestinal
Epithelial Cells. Front. Vet. Sci. 2021, 8, 677897. [CrossRef] [PubMed]
Dubreuil, J.D.; Isaacson, R.E.; Schifferli, D.M. Animal Enterotoxigenic Escherichia coli. EcoSal Plus 2016, 7, 10. [CrossRef]
Kobayashi, H.; Albarracin, L.; Sato, N.; Kanmani, P.; Kober, A.; Ikeda-Ohtsubo, W.; Suda, Y.; Nochi, T.; Aso, H.; Makino, S.; et al.
Modulation of Porcine Intestinal Epitheliocytes Immunetranscriptome Response by Lactobacillus jensenii TL2937. Benef. Microbes
2016, 7, 769–782. [CrossRef]
Hu, M.M.; Shu, H.B. Cytoplasmic Mechanisms of Recognition and Defense of Microbial Nucleic Acids. Annu. Rev. Cell Dev. Biol.
2018, 34, 357–379. [CrossRef]
Afonina, I.S.; Zhong, Z.; Karin, M.; Beyaert, R. Limiting Inflammation-the Negative Regulation of NF-KB and the NLRP3Inflammasome. Nat. Immunol. 2017, 18, 861–869. [CrossRef]
Zhou, M.; Xu, W.; Wang, J.; Yan, J.; Shi, Y.; Zhang, C.; Ge, W.; Wu, J.; Du, P.; Chen, Y. Boosting MTOR-Dependent Autophagy via
Upstream TLR4-MyD88-MAPK Signalling and Downstream NF-KB Pathway Quenches Intestinal Inflammation and Oxidative
Stress Injury. EBioMedicine 2018, 35, 345–360. [CrossRef]
Brubaker, J.; Zhang, X.; Bourgeois, A.L.; Harro, C.; Sack, D.A.; Chakraborty, S. Intestinal and Systemic Inflammation Induced
by Symptomatic and Asymptomatic Enterotoxigenic E. coli Infection and Impact on Intestinal Colonization and ETEC Specific
Immune Responses in an Experimental Human Challenge Model. Gut Microbes 2021, 13, 1–13. [CrossRef]

Microorganisms 2023, 11, 63

10.
11.
12.
13.
14.
15.
16.
17.

18.

19.
20.

21.

22.
23.

24.
25.
26.

27.

28.

29.

30.

31.
32.
33.

15 of 17

Jiang, F.; Wu, Z.; Zheng, Y.; Frana, T.S.; Sahin, O.; Zhang, Q.; Li, G. Genotypes and Antimicrobial Susceptibility Profiles ofHemolytic Escherichia coli from Diarrheic Piglets. Foodborne Pathog. Dis. 2019, 16, 94–103. [CrossRef]de Lagarde, M.; Vanier, G.; Arsenault, J.; Fairbrother, J.M. High Risk Clone: A Proposal of Criteria Adapted to the One HealthContext with Application to Enterotoxigenic Escherichia coli in the Pig Population. Antibiotics 2021, 10, 244. [CrossRef] [PubMed]
Dubreuil, J.D. Enterotoxigenic Escherichia coli and Probiotics in Swine: What the Bleep Do We Know? Biosci. Microbiota Food Health2017, 36, 75–90. [CrossRef] [PubMed]
Shu, Q.; Gill, H.S. A dietary probiotic (Bifidobacterium lactis HN019) reduces the severity of Escherichia coli O157:H7 infection in
mice. Med. Microbiol. Immunol. 2001, 189, 147–152. [CrossRef] [PubMed]
Shu, Q.; Gill, H.S. Immune protection mediated by the probiotic Lactobacillus rhamnosus HN001 (DR20) against Escherichia coli
O157:H7 infection in mice. FEMS Immunol. Med. Microbiol. 2002, 34, 59–64. [CrossRef]
Shu, Q.; Qu, F.; Gill, H.S. Probiotic treatment using Bifidobacterium lactis HN019 reduces weanling diarrhea associated withrotavirus and Escherichia coli infection in a piglet model. J. Pediatr. Gastroenterol. Nutr. 2001, 33, 171–177. [CrossRef]
Sun, Y.; Duarte, M.E.; Kim, S.W. Dietary inclusion of multispecies probiotics to reduce the severity of post-weaning diarrhea
caused by Escherichia coli F18+ in pigs. Anim. Nutr. 2021, 7, 326–333. [CrossRef]
Daudelin, J.F.; Lessard, M.; Beaudoin, F.; Nadeau, E.; Bissonnette, N.; Boutin, Y.; Brousseau, J.P.; Lauzon, K.; Fairbrother, J.M.
Administration of probiotics influences F4 (K88)-positive enterotoxigenic Escherichia coli attachment and intestinal cytokine
expression in weaned pigs. Veter Res. 2011, 42, 69. [CrossRef]
Garcia-Castillo, V.; Komatsu, R.; Clua, P.; Indo, Y.; Takagi, M.; Salva, S.; Islam, M.A.; Alvarez, S.; Takahashi, H.; Garcia-Cancino, A.;et al. Evaluation of the Immunomodulatory Activities of the Probiotic Strain Lactobacillus fermentum UCO-979C. Front. Immunol.
2019, 10, 1376. [CrossRef]
Villena, J.; Kitazawa, H. Modulation of Intestinal TLR4-Inflammatory Signaling Pathways by Probiotic Microorganisms: Lessons
Learned from Lactobacillus jensenii TL2937. Front. Immunol. 2014, 4, 512. [CrossRef]
Moue, M.; Tohno, M.; Shimazu, T.; Kido, T.; Aso, H.; Saito, T.; Kitazawa, H. Toll-like Receptor 4 and Cytokine Expression Involved
in Functional Immune Response in an Originally Established Porcine Intestinal Epitheliocyte Cell Line. Biochim. Biophys. Acta
Gen. Subj. 2008, 1780, 134–144. [CrossRef]
Shimazu, T.; Villena, J.; Tohno, M.; Fujie, H.; Hosoya, S.; Shimosato, T.; Aso, H.; Suda, Y.; Kawai, Y.; Saito, T.; et al. Immunobiotic
Lactobacillus jensenii Elicits Anti-Inflammatory Activity in Porcine Intestinal Epithelial Cells by Modulating Negative Regulatorsof the Toll-like Receptor Signaling Pathway. Infect. Immun. 2012, 80, 276–288. [CrossRef] [PubMed]de Vries, M.C.; Vaughan, E.E.; Kleerebezem, M.; de Vos, W.M. Lactobacillus plantarum-Survival, Functional and Potential Probiotic
Properties in the Human Intestinal Tract. Int. Dairy J. 2006, 16, 1018–1028. [CrossRef]
Siezen, R.J.; Tzeneva, V.A.; Castioni, A.; Wels, M.; Phan, H.T.K.; Rademaker, J.L.W.; Starrenburg, M.J.C.; Kleerebezem, M.; vanHylckama Vlieg, J.E.T. Phenotypic and Genomic Diversity of Lactobacillus plantarum Strains Isolated from Various Environmental
Niches. Environ. Microbiol. 2010, 12, 758–773. [CrossRef] [PubMed]
Villena, J.; Li, C.; Vizoso-Pinto, M.G.; Sacur, J.; Ren, L.; Kitazawa, H. Lactiplantibacillus plantarum as a Potential Adjuvant andDelivery System for the Development of SARS-CoV-2 Oral Vaccines. Microorganisms 2021, 9, 683. [CrossRef] [PubMed]
Behera, S.S.; Ray, R.C.; Zdolec, N. Lactobacillus plantarum with Functional Properties: An Approach to Increase Safety and
Shelf-Life of Fermented Foods. Biomed Res. Int 2018, 2018, 9361614. [CrossRef]
Albarracin, L.; Garcia-Castillo, V.; Masumizu, Y.; Indo, Y.; Islam, M.A.; Suda, Y.; Garcia-Cancino, A.; Aso, H.; Takahashi, H.;
Kitazawa, H.; et al. Efficient Selection of New Immunobiotic Strains With Antiviral Effects in Local and Distal Mucosal Sites byUsing Porcine Intestinal Epitheliocytes. Front. Immunol. 2020, 11, 543. [CrossRef]
Mizuno, H.; Arce, L.; Tomotsune, K.; Albarracin, L.; Funabashi, R.; Vera, D.; Islam, M.A.; Vizoso-Pinto, M.G.; Takahashi, H.;
Sasaki, Y.; et al. Lipoteichoic Acid Is Involved in the Ability of the Immunobiotic Strain Lactobacillus plantarum CRL1506 to
Modulate the Intestinal Antiviral Innate Immunity Triggered by TLR3 Activation. Front. Immunol. 2020, 11, 571. [CrossRef]
Islam, M.A.; Albarracin, L.; Tomokiyo, M.; Valdez, J.C.; Sacur, J.; Vizoso-Pinto, M.G.; Andrade, B.G.N.; Cuadrat, R.R.C.; Kitazawa,
H.; Villena, J. Immunobiotic Lactobacilli Improve Resistance of Respiratory Epithelial Cells to SARS-CoV-2 Infection. Pathogens
2021, 10, 1197. [CrossRef]
Tada, A.; Zelaya, H.; Clua, P.; Salva, S.; Alvarez, S.; Kitazawa, H.; Villena, J. Immunobiotic Lactobacillus Strains Reduce Small
Intestinal Injury Induced by Intraepithelial Lymphocytes after Toll-like Receptor 3 Activation. Inflamm. Res. 2016, 65, 771–783.
[CrossRef]
Albarracin, L.; Tonetti, F.R.; Fukuyama, K.; Suda, Y.; Zhou, B.; Baillo, A.A.; Fadda, S.; Saavedra, L.; Kurata, S.; Hebert, E.M.; et al.
Genomic Characterization of Lactiplantibacillus plantarum Strains Possessing Differential Antiviral Immunomodulatory Activities.
Bacteria 2022, 1, 136–160. [CrossRef]
Fadda, S.; Vildoza, M.J.; Vignolo, G. The acidogenic metabolism of Lactobacillus plantarum CRL 681 improves sarcoplasmic proteinhydrolysis during meat fermentation. J. Muscle Foods 2010, 21, 545–556. [CrossRef]
Fadda, S.; Chambon, C.; Champomier-Vergès, M.C.; Talon, R.; Vignolo, G. Lactobacillus Role during Conditioning of Refrigerated
and Vacuum-Packaged Argentinean Meat. Meat Sci. 2008, 79, 603–610. [CrossRef] [PubMed]de Almeida, M.A.; Saldaña, E.; da Silva Pinto, J.S.; Palacios, J.; Contreras-Castillo, C.J.; Sentandreu, M.A.; Fadda, S.G. A Peptidomic
Approach of Meat Protein Degradation in a Low-Sodium Fermented Sausage Model Using Autochthonous Starter Cultures.
Food Res. Int. 2018, 109, 368–379. [CrossRef] [PubMed]

Microorganisms 2023, 11, 63

34.
35.

36.

37.

38.
39.
40.
41.

42.
43.
44.
45.
46.

47.
48.
49.

50.

51.

52.

53.

54.
55.

56.

16 of 17

Fadda, S.; López, C.; Vignolo, G. Role of Lactic Acid Bacteria during Meat Conditioning and Fermentation: Peptides Generated as
Sensorial and Hygienic Biomarkers. Meat Sci. 2010, 86, 66–79. [CrossRef] [PubMed]
Orihuel, A.; Terán, L.; Renaut, J.; Vignolo, G.M.; de Almeida, A.M.; Saavedra, M.L.; Fadda, S. Differential Proteomic Analysis of
Lactic Acid Bacteria- Escherichia coli O157:H7 Interaction and Its Contribution to Bioprotection Strategies in Meat. Front. Microbiol.
2018, 9, 1083. [CrossRef]
Tomosada, Y.; Villena, J.; Murata, K.; Chiba, E.; Shimazu, T.; Aso, H.; Iwabuchi, N.; Xiao, J.Z.; Saito, T.; Kitazawa, H. Immunoregulatory Effect of Bifidobacteria Strains in Porcine Intestinal Epithelial Cells through Modulation of Ubiquitin-Editing Enzyme A20
Expression. PLoS One 2013, 8, e59259. [CrossRef] [PubMed]
Indo, Y.; Kitahara, S.; Tomokiyo, M.; Araki, S.; Islam, M.A.; Zhou, B.; Albarracin, L.; Miyazaki, A.; Ikeda-Ohtsubo, W.; Nochi, T.;et al. Ligilactobacillus Salivarius Strains Isolated From the Porcine Gut Modulate Innate Immune Responses in Epithelial Cells andImprove Protection Against Intestinal Viral-Bacterial Superinfection. Front. Immunol. 2021, 12, 1. [CrossRef]
Marranzino, G.; Villena, J.; Salva, S.; Alvarez, S. Stimulation of Macrophages by Immunobiotic Lactobacillus Strains: Influence
beyond the Intestinal Tract. Microbiol. Immunol. 2012, 56, 771–781. [CrossRef]
Adetoye, A.; Pinloche, E.; Adeniyi, B.A.; Ayeni, F.A. Characterization and Anti-Salmonella Activities of Lactic Acid Bacteria
Isolated from Cattle Faeces. BMC Microbiol. 2018, 18, 96. [CrossRef]
Markowiak, P.; Ślizewska, K. The Role of Probiotics, Prebiotics and Synbiotics in Animal Nutrition. Gut Pathog. 2018, 10, 21.
[CrossRef]
Chaillou, S.; Champomier-Vergès, M.C.; Cornet, M.; Crutz-Le Coq, A.M.; Dudez, A.M.; Martin, V.; Beaufils, S.; Darbon-Rongère,E.; Bossy, R.; Loux, V.; et al. The Complete Genome Sequence of the Meat-Borne Lactic Acid Bacterium Lactobacillus sakei 23K. Nat.
Biotechnol. 2005, 23, 1527–1533. [CrossRef]
Hebert, E.M.; Saavedra, L.; Taranto, M.P.; Mozzi, F.; Magni, C.; Nader, M.E.F.; de Valdez, G.F.; Sesma, F.; Vignolo, G.; Raya, R.R.
Genome Sequence of the Bacteriocin-Producing Lactobacillus curvatus Strain CRL705. J. Bacteriol. 2012, 194, 538–539. [CrossRef]
Corsetti, A.; Valmorri, S. Lactic Acid Bacteria|Lactobacillus spp.: Lactobacillus plantarum. Encycl. Dairy Sci. Second. Ed. 2011, 3,
111–118. [CrossRef]
Vignolo, G.M.; de Ruiz Holgado, A.P.; Oliver, G. Acid Production and Proteolytic Activity of Lactobacillus Strains Isolated fromDry Sausages. J. Food Prot. 1988, 51, 481–484. [CrossRef]
Fadda, S.; Vignolo, G.; Oliver, G. Tyramine Degradation and Tyramine/Histamine Production by Lactic Acid Bacteria and Kocuria
Strains. Biotechnol. Lett. 2001, 23, 2015–2019. [CrossRef]
Albarracin, L.; Kobayashi, H.; Iida, H.; Sato, N.; Nochi, T.; Aso, H.; Salva, S.; Alvarez, S.; Kitazawa, H.; Villena, J. Transcriptomic
Analysis of the Innate Antiviral Immune Response in Porcine Intestinal Epithelial Cells: Influence of Immunobiotic Lactobacilli.
Front. Immunol. 2017, 8, 57. [CrossRef]
Maldonado Galdeano, C.; de Moreno De Leblanc, A.; Vinderola, G.; Bibas Bonet, M.E.; Perdigón, G. Proposed Model: Mechanismsof Immunomodulation Induced by Probiotic Bacteria. Clin. Vaccine Immunol. 2007, 14, 485–492. [CrossRef]
Salva, S.; Villena, J.; Alvarez, S. Immunomodulatory Activity of Lactobacillus rhamnosus Strains Isolated from Goat Milk: Impact
on Intestinal and Respiratory Infections. Int. J. Food Microbiol. 2010, 141, 82–89. [CrossRef]
Villena, J.; Chiba, E.; Vizoso-Pinto, M.G.; Tomosada, Y.; Takahashi, T.; Ishizuka, T.; Aso, H.; Salva, S.; Alvarez, S.; Kitazawa, H.
Immunobiotic Lactobacillus rhamnosus Strains Differentially Modulate Antiviral Immune Response in Porcine Intestinal Epithelialand Antigen Presenting Cells. BMC Microbiol. 2014, 14, 126. [CrossRef]
Grivennikov, S.; Karin, E.; Terzic, J.; Mucida, D.; Yu, G.Y.; Vallabhapurapu, S.; Scheller, J.; Rose-John, S.; Cheroutre, H.; Eckmann,
L.; et al. IL-6 and Stat3 Are Required for Survival of Intestinal Epithelial Cells and Development of Colitis-Associated Cancer.
Cancer Cell 2009, 15, 103–113. [CrossRef]
Li, Y.Y.; Ishihara, S.; Aziz, M.M.; Oka, A.; Kusunoki, R.; Tada, Y.; Yuki, T.; Amano, Y.; Ansary, M.U.; Kinoshita, Y. Autophagy Is
Required for Toll-like Receptor-Mediated Interleukin-8 Production in Intestinal Epithelial Cells. Int. J. Mol. Med. 2011, 27, 337–344.
[CrossRef]
Elean, M.; Albarracin, L.; Fukuyama, K.; Zhou, B.; Tomokiyo, M.; Kitahara, S.; Araki, S.; Suda, Y.; Saavedra, L.; Villena, J.;et al. Lactobacillus delbrueckii CRL 581 Differentially Modulates TLR3-Triggered Antiviral Innate Immune Response in Intestinal
Epithelial Cells and Macrophages. Microorganisms 2021, 9, 2449. [CrossRef] [PubMed]
Roselli, M.; Finamore, A.; Britti, M.S.; Mengheri, E. Probiotic Bacteria Bifidobacterium animalis MB5 and Lactobacillus rhamnosus GG
Protect Intestinal Caco-2 Cells from the Inflammation-Associated Response Induced by Enterotoxigenic Escherichia coli K88. Br. J.
Nutr. 2006, 95, 1177–1184. [CrossRef] [PubMed]
Tian, Z.; Liu, X.; Dai, R.; Xiao, Y.; Wang, X.; Bi, D.; Shi, D. Enterococcus faecium HDRsEf1 Protects the Intestinal Epithelium andAttenuates ETEC-Induced IL-8 Secretion in Enterocytes. Mediat. Inflamm. 2016, 6, 7474306. [CrossRef]
Roselli, M.; Finamore, A.; Britti, M.S.; Konstantinov, S.R.; Smidt, H.; de Vos, W.M.; Mengheri, E. The Novel Porcine Lactobacillus
sobrius Strain Protects Intestinal Cells from Enterotoxigenic Escherichia coli K88 Infection and Prevents Membrane Barrier Damage.
J. Nutr. 2007, 137, 2709–2716. [CrossRef] [PubMed]
Yang, J.; Qiu, Y.; Hu, S.; Zhu, C.; Wang, L.; Wen, X.; Yang, X.; Jiang, Z. Lactobacillus plantarum Inhibited the Inflammatory Response
Induced by Enterotoxigenic Escherichia coli K88 via Modulating MAPK and NF-KB Signalling in Intestinal Porcine Epithelial Cells.
J. Appl. Microbiol. 2021, 130, 1684–1694. [CrossRef] [PubMed]

Microorganisms 2023, 11, 63

57.

58.

59.
60.

61.
62.

63.
64.
65.

17 of 17

Kessel, A.; Toubi, E.; Pavlotzky, E.; Mogilner, J.; Coran, A.G.; Lurie, M.; Karry, R.; Sukhotnik, I. Treatment with Glutamine
Is Associated with Down-Regulation of Toll-like Receptor-4 and Myeloid Differentiation Factor 88 Expression and Decreasein Intestinal Mucosal Injury Caused by Lipopolysaccharide Endotoxaemia in a Rat. Clin. Exp. Immunol. 2008, 151, 341–347.
[CrossRef]
Yue, Y.; He, Z.; Zhou, Y.; Ross, R.P.; Stanton, C.; Zhao, J.; Zhang, H.; Yang, B.; Chen, W. Lactobacillus plantarum Relieves Diarrhea
Caused by Enterotoxin-Producing Escherichia coli through Inflammation Modulation and Gut Microbiota Regulation. Food Funct.
2020, 11, 10362–10374. [CrossRef]
Hubbard, L.L.N.; Moore, B.B. IRAK-M Regulation and Function in Host Defense and Immune Homeostasis. Infect. Dis. Rep. 2010,
2, 22–29. [CrossRef]
Escoll, P.; del Fresno, C.; García, L.; Vallés, G.; Lendínez, M.J.; Arnalich, F.; López-Collazo, E. Rapid up-Regulation of IRAK-M
Expression Following a Second Endotoxin Challenge in Human Monocytes and in Monocytes Isolated from Septic Patients.
Biochem. Biophys. Res. Commun. 2003, 311, 465–472. [CrossRef]
Ning, S.; Pagano, J.S. The A20 Deubiquitinase Activity Negatively Regulates LMP1 Activation of IRF7. J. Virol. 2010, 84, 6130–6138.
[CrossRef]
Vereecke, L.; Sze, M.; Mc Guire, C.; Rogiers, B.; Chu, Y.; Schmidt-Supprian, M.; Pasparakis, M.; Beyaert, R.; van Loo, G. EnterocyteSpecific A20 Deficiency Sensitizes to Tumor Necrosis Factor-Induced Toxicity and Experimental Colitis. J. Exp. Med. 2010, 207,
1513–1523. [CrossRef]
Wessells, J.; Baer, M.; Young, H.A.; Claudio, E.; Brown, K.; Siebenlist, U.; Johnson, P.F. BCL-3 and NF-KappaB P50 Attenuate
Lipopolysaccharide-Induced Inflammatory Responses in Macrophages. J. Biol. Chem. 2004, 279, 49995–50003. [CrossRef]
Wang, J.; Ford, H.R.; Grishin, A.V. NF-KappaB-Mediated Expression of MAPK Phosphatase-1 Is an Early Step in Desensitization
to TLR Ligands in Enterocytes. Mucosal Immunol. 2010, 3, 523–534. [CrossRef]
Stephens, M.; von der Weid, P.Y. Lipopolysaccharides Modulate Intestinal Epithelial Permeability and Inflammation in aSpecies-Specific Manner. Gut Microbes 2020, 11, 421–432. [CrossRef]

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