In Vitro Evaluation of the Most Active Probiotic Strains Able to Improve the Intestinal Barrier Functions and to Prevent Inflammatory Diseases of the Gastrointestinal System
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Cultures
2.2. Bacterial Strains
2.3. Cell Treatment
2.4. Real-Time PCR
2.5. ELISA Assay
2.6. Adhesion and Invasiveness Assay
2.7. Statistical Analysis
3. Results
3.1. Regulation of TJ Expression
3.2. Induction of Innate Immune Response
3.3. Anti-Inflammarory Activity of Lactobacillus spp.
3.4. Activity of Lactobacilli against S. Typhimurium and EIEC Adhesion and Invasion
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Soderholm, A.T.; Pedicord, V.A. Intestinal epithelial cells: At the interface of the microbiota and mucosal immunity. Immunology 2019, 158, 267–280. [Google Scholar] [CrossRef] [Green Version]
- Wittkopf, N.; Neurath, M.F.; Becker, C. Immune-epithelial crosstalk at the intestinal surface. J. Gastroenterol. 2014, 49, 375–387. [Google Scholar] [CrossRef] [PubMed]
- Fusco, A.; Savio, V.; Cammarota, M.; Alfano, A.; Schiraldi, C.; Donnarumma, G. Beta-Defensin-2 and Beta-Defensin-3 Reduce Intestinal Damage Caused by Salmonella typhimurium Modulating the Expression of Cytokines and Enhancing the Probiotic Activity of Enterococcus faecium. J. Immunol. Res. 2017, 2017, 6976935. [Google Scholar] [CrossRef] [Green Version]
- Vancamelbeke, M.; Vermeire, S. The intestinal barrier: A fundamental role in health and disease. Expert Rev. Gastroenterol. Hepatol. 2017, 11, 821–834. [Google Scholar] [CrossRef]
- Grondin, J.A.; Kwon, Y.H.; Far, P.M.; Haq, S.; Khan, W.I. Mucins in Intestinal Mucosal Defense and Inflammation: Learning from Clinical and Experimental Studies. Front. Immunol. 2020, 11, 2054. [Google Scholar] [CrossRef]
- Fusco, A.; Savio, V.; Donniacuo, M.; Perfetto, B.; Donnarumma, G. Antimicrobial Peptides Human Beta-Defensin-2 and -3 Protect the Gut During Candida albicans Infections Enhancing the Intestinal Barrier Integrity: In Vitro Study. Front Cell Infect Microbiol. 2021, 11, 666900. [Google Scholar] [CrossRef]
- Barbara, G.; Barbaro, M.R.; Fuschi, D.; Palombo, M.; Falangone, F.; Cremon, C.; Marasco, G.; Stanghellini, V. Inflammatory and Microbiota-Related Regulation of the Intestinal Epithelial Barrier. Front. Nutr. 2021, 8, 718356. [Google Scholar] [CrossRef]
- Ghosh, S.S.; Wang, J.; Yannie, P.J.; Ghosh, S. Intestinal Barrier Dysfunction, LPS Translocation, and Disease Development. J. Endocr. Soc. 2020, 4, bvz039. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Stolfi, C.; Maresca, C.; Monteleone, G.; Laudisi, F. Implication of Intestinal Barrier Dysfunction in Gut Dysbiosis and Diseases. Biomedicines 2022, 10, 289. [Google Scholar] [CrossRef]
- Lee, S.H.; Kwon, J.E.; Cho, M.L. Immunological pathogenesis of inflammatory bowel disease. Intest. Res. 2018, 16, 26–42. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Qiu, P.; Ishimoto, T.; Fu, L.; Zhang, J.; Zhang, Z.; Liu, Y. The Gut Microbiota in Inflammatory Bowel Disease. Front. Cell. Infect. Microbiol. 2022, 12, 733992. [Google Scholar] [CrossRef]
- Schumann, M.; Siegmund, B.; Schulzke, J.D.; Fromm, M. Celiac Disease: Role of the Epithelial Barrier. Cell Mol. Gastroenterol. Hepatol. 2017, 3, 150–162. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Genua, F.; Raghunathan, V.; Jenab, M.; Gallagher, W.M.; Hughes, D.J. The Role of Gut Barrier Dysfunction and Microbiome Dysbiosis in Colorectal Cancer Development. Front. Oncol. 2021, 11, 626349. [Google Scholar] [CrossRef]
- Popa, G.L.; Papa, M.I. Salmonella spp. Infection—A continuous threat worldwide. Germs 2021, 11, 88–96. [Google Scholar] [CrossRef] [PubMed]
- Fusco, A.; Savio, V.; Perfetto, B.; Mattina, R.; Donnarumma, G. Antimicrobial peptide human β-defensin-2 improves in vitro cellular viability and reduces pro-inflammatory effects induced by enteroinvasive Escherichia coli in Caco-2 cells by inhibiting invasion and virulence factors’ expression. Front. Cell Infect. Microbiol. 2022, 12, 1009415. [Google Scholar] [CrossRef] [PubMed]
- Müller, J.; Spriewald, S.; Stecher, B.; Stadler, E.; Fuchs, T.M. Evolutionary Stability of Salmonella Competition with the Gut Microbiota: How the Environment Fosters Heterogeneity in Exploitative and Interference Competition. J. Mol. Biol. 2019, 431, 4732–4748. [Google Scholar] [CrossRef]
- Jiang, L.; Wang, P.; Song, X.; Zhang, H.; Ma, S.; Wang, J.; Li, W.; Lv, R.; Liu, X.; Ma, S.; et al. Salmonella Typhimurium reprograms macrophage metabolism via T3SS effector SopE2 to promote intracellular replication and virulence. Nat. Commun. 2021, 12, 879. [Google Scholar] [CrossRef] [PubMed]
- Wang, K.C.; Huang, C.H.; Chang, P.R.; Huang, M.T.; Fang, S.B. Role of wzxE in Salmonella Typhimurium lipopolysaccharide biosynthesis and interleukin-8 secretion regulation in human intestinal epithelial cells. Microbiol. Res. 2020, 238, 126502. [Google Scholar] [CrossRef] [PubMed]
- Wemyss, M.A.; Pearson, J.S. Host Cell Death Responses to Non-typhoidal Salmonella Infection. Front. Immunol. 2019, 10, 1758. [Google Scholar] [CrossRef]
- Marchello, C.S.; Birkhold, M.; Crump, J.A. Vacc-iNTS Consortium Collaborators. Complications and mortality of non-typhoidal Salmonella invasive disease: A global systematic review and meta-analysis. Lancet Infect. Dis. 2022, 22, 692–705. [Google Scholar] [CrossRef]
- Mohammadzadeh, M.; Goudarzi, H.; Dabiri, H.; Fallah, F. Molecular detection of lactose fermenting enteroinvasive Escherichia coli from patients with diarrhea in Tehran-Iran. Iran. J. Microbiol. 2015, 7, 198. [Google Scholar]
- Pasqua, M.; Michelacci, V.; Di Martino, M.L.; Tozzoli, R.; Grossi, M.; Colonna, B.; Morabito, S.; Prosseda, G. The Intriguing Evolutionary Journey of Enteroinvasive E. coli (EIEC) toward Pathogenicity. Front. Microbiol. 2017, 8, 2390. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Mazziotta, C.; Tognon, M.; Martini, F.; Torreggiani, E.; Rotondo, J.C. Probiotics Mechanism of Action on Immune Cells and Beneficial Effects on Human Health. Cells 2023, 12, 184. [Google Scholar] [CrossRef] [PubMed]
- Boirivant, M.; Strober, W. The mechanism of action of probiotics. Curr. Opin. Gastroenterol. 2007, 23, 679–692. [Google Scholar] [CrossRef]
- Binda, S.; Hill, C.; Johansen, E.; Obis, D.; Pot, B.; Sanders, M.E.; Tremblay, A.; Ouwehand, A.C. Criteria to Qualify Microorganisms as “Probiotic” in Foods and Dietary Supplements. Front. Microbiol. 2020, 11, 1662. [Google Scholar] [CrossRef] [PubMed]
- Damián, M.R.; Cortes-Perez, N.G.; Quintana, E.T.; Ortiz-Moreno, A.; Garfias Noguez, C.; Cruceño-Casarrubias, C.E.; Sánchez Pardo, M.E.; Bermúdez-Humarán, L.G. Functional Foods, Nutraceuticals and Probiotics: A Focus on Human Health. Microorganisms 2022, 10, 1065. [Google Scholar] [CrossRef] [PubMed]
- Dempsey, E.; Corr, S.C. Lactobacillus spp. for Gastrointestinal Health: Current and Future Perspectives. Front. Immunol. 2022, 13, 840245. [Google Scholar] [CrossRef]
- Duar, R.M.; Lin, X.B.; Zheng, J.; Martino, M.E.; Grenier, T.; Pérez-Muñoz, M.E.; Leulier, F.; Gänzle, M.; Walter, J. Lifestyles in transition: Evolution and natural history of the genus Lactobacillus. FEMS Microbiol. Rev. 2017, 41 (Suppl. 1), S27–S48. [Google Scholar] [CrossRef] [Green Version]
- Plaza-Diaz, J.; Ruiz-Ojeda, F.J.; Gil-Campos, M.; Gil, A. Mechanisms of Action of Probiotics. Adv. Nutr. 2019, 10 (Suppl. 1), S49–S66. [Google Scholar] [CrossRef] [Green Version]
- 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. [Google Scholar] [CrossRef] [Green Version]
- Meng, Y.; Li, B.; Jin, D.; Zhan, M.; Lu, J.; Huo, G. Immunomodulatory activity of Lactobacillus plantarum KLDS1.0318 in cyclophosphamide-treated mice. Food Nutr Res. 2018, 62. [Google Scholar] [CrossRef] [Green Version]
- D’ambrosio, S.; Ventrone, M.; Fusco, A.; Casillo, A.; Dabous, A.; Cammarota, M.; Corsaro, M.M.; Donnarumma, G.; Schiraldi, C.; Cimini, D. Limosilactobacillus fermentum from buffalo milk is suitable for potential biotechnological process development and inhibits Helicobacter pylori in a gastric epithelial cell model. Biotechnol. Rep. 2022, 34, e00732. [Google Scholar] [CrossRef]
- Wells, J.M. Immunomodulatory mechanisms of lactobacilli. Microb. Cell Fact. 2011, 10 (Suppl. 1), S17. [Google Scholar] [CrossRef] [Green Version]
- Cieślik, M.; Bagińska, N.; Górski, A.; Jończyk-Matysiak, E. Human β-Defensin 2 and Its Postulated Role in Modulation of the Immune Response. Cells 2021, 10, 2991. [Google Scholar] [CrossRef] [PubMed]
- Gubatan, J.; Holman, D.R.; Puntasecca, C.J.; Polevoi, D.; Rubin, S.J.; Rogalla, S. Antimicrobial peptides and the gut microbiome in inflammatory bowel disease. World J. Gastroenterol. 2021, 27, 7402–7422. [Google Scholar] [CrossRef] [PubMed]
- Qin, D.; Ma, Y.; Wang, Y.; Hou, X.; Yu, L. Contribution of Lactobacilli on Intestinal Mucosal Barrier and Diseases: Perspectives and Challenges of Lactobacillus casei. Life 2022, 12, 1910. [Google Scholar] [CrossRef] [PubMed]
- Herath, M.; Hosie, S.; Bornstein, J.C.; Franks, A.E.; Hill-Yardin, E.L. The Role of the Gastrointestinal Mucus System in Intestinal Homeostasis: Implications for Neurological Disorders. Front. Cell. Infect. Microbiol. 2020, 10, 248. [Google Scholar] [CrossRef] [PubMed]
- Nie, S.; Yuan, Y. The Role of Gastric Mucosal Immunity in Gastric Diseases. J. Immunol. Res. 2020, 2020, 7927054. [Google Scholar] [CrossRef]
- Okumura, R.; Takeda, K. Maintenance of intestinal homeostasis by mucosal barriers. Inflamm. Regen. 2018, 38, 5. [Google Scholar] [CrossRef]
- Ayivi, R.D.; Gyawali, R.; Krastanov, A.; Aljaloud, S.O.; Worku, M.; Tahergorabi, R.; Silva, R.C.D.; Ibrahim, S.A. Lactic Acid Bacteria: Food Safety and Human Health Applications. Dairy 2020, 1, 202–232. [Google Scholar] [CrossRef]
- Rossi, F.; Amadoro, C.; Colavita, G. Members of the Lactobacillus Genus Complex (LGC) as Opportunistic Pathogens: A Review. Microorganisms 2019, 7, 126. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Azad, M.A.K.; Sarker, M.; Li, T.; Yin, J. Probiotic Species in the Modulation of Gut Microbiota: An Overview. BioMed Res. Int. 2018, 2018, 9478630. [Google Scholar] [CrossRef] [Green Version]
- Rastogi, S.; Singh, A. Gut microbiome and human health: Exploring how the probiotic genus Lactobacillus modulate immune responses. Front. Pharmacol. 2022, 13, 1042189. [Google Scholar] [CrossRef] [PubMed]
- Chen, Y.; Xu, J.; Chen, Y. Regulation of Neurotransmitters by the Gut Microbiota and Effects on Cognition in Neurological Disorders. Nutrients 2021, 13, 2099. [Google Scholar] [CrossRef] [PubMed]
- Angelin, J.; Kavitha, M. Exopolysaccharides from probiotic bacteria and their health potential. Int. J. Biol. Macromol. 2020, 162, 853–865. [Google Scholar] [CrossRef] [PubMed]
- Kumar, A.S.; Mody, K.; Jha, B. Bacterial exopolysaccharides—A perception. J. Basic Microbiol. 2007, 47, 103–117. [Google Scholar] [CrossRef]
- El Houari, A.; Ecale, F.; Mercier, A.; Crapart, S.; Laparre, J.; Soulard, B.; Ramnath, M.; Berjeaud, J.-M.; Rodier, M.-H.; Crépin, A. Development of an in vitro Model of Human Gut Microbiota for Screening the Reciprocal Interactions with Antibiotics, Drugs, and Xenobiotics. Front. Microbiol. 2022, 13, 828359. [Google Scholar] [CrossRef] [PubMed]
- Mahler, G.J.; Shuler, M.L.; Glahn, R.P. Characterization of Caco-2 and HT29-MTX cocultures in an in vitro digestion/cell culture model used to predict iron bioavailability. J. Nutr. Biochem. 2009, 20, 494–502. [Google Scholar] [CrossRef] [PubMed]
- Cimini, D.; D’ambrosio, S.; Stellavato, A.; Fusco, A.; Corsaro, M.M.; Dabous, A.; Casillo, A.; Donnarumma, G.; Giori, A.M.; Schiraldi, C. Optimization of growth of Levilactobacillus brevis SP 48 and in vitro evaluation of the effect of viable cells and high molecular weight potential postbiotics on Helicobacter pylori. Front. Bioeng. Biotechnol. 2022, 10, 1007004. [Google Scholar] [CrossRef]
- Zarłok, K. Lactobacillus fermentum CECT5716—Probiotic from human milk with interesting properties. Wiad. Lek. 2016, 69, 271–275. [Google Scholar]
- Verdenelli, M.C.; Ghelfi, F.; Silvi, S.; Orpianesi, C.; Cecchini, C.; Cresci, A. Probiotic properties of Lactobacillus rhamnosus and Lactobacillus paracasei isolated from human faeces. Eur. J. Nutr. 2009, 48, 355–363. [Google Scholar] [CrossRef] [PubMed]
- Verdenelli, M.C.; Silvi, S.; Cecchini, C.; Orpianesi, C.; Cresci, A. Influence of a combination of two potential probiotic strains, Lactobacillus rhamnosus IMC 501® and Lactobacillus paracasei IMC 502® on bowel habits of healthy adults. Lett. Appl. Microbiol. 2011, 52, 596–602. [Google Scholar] [CrossRef] [PubMed]
- Mu, Q.; Kirby, J.; Reilly, C.M.; Luo, X.M. Leaky gut as a danger signal for autoimmune diseases. Front. Immunol. 2017, 8, 598. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Mu, Q.; Tavella, V.J.; Luo, X.M. Role of Lactobacillus reuteri in Human Health and Diseases. Front. Microbiol. 2018, 9, 757. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Pourmirzaiee, M.A.; Famouri, F.; Moazeni, W.; Hassanzadeh, A.; Hajihashemi, M. The efficacy of the prenatal administration of Lactobacillus reuteri LR92 DSM 26866 on the prevention of infantile colic: A randomized control trial. Eur. J. Pediatr. 2020, 179, 1619–1626. [Google Scholar] [CrossRef]
- Islam, S.U. Clinical Uses of Probiotics. Medicine 2016, 95, e2658. [Google Scholar] [CrossRef]
Gene | Primer Sequences | Conditions | Product Size (bp) |
---|---|---|---|
IL-6 | 5′-ATGAACTCCTTCTCCACAAGCGC-3′ 5′-GAAGAGCCCTCAGGCTGGACTG-3′ | 5″ at 95 °C, 13″ at 56 °C, 25″ at 72 °C for 40 cycles | 628 |
IL-8 | 5′-ATGACTTCCAAGCTGGCCGTG-3′ 5′-TGAATTCTCAGCCCTCTTCAAAAACTTCTC-3′ | 5″ at 94 °C, 6″ at 55 °C, 12″ at 72 °C for 40 cycles | 297 |
IL-1α | 5′-CATGTCAAATTTCACTGCTTCATCC-3′ 5′-GTCTCTGAATCAGAAATCCTTCTATC-3′ | 5″ at 95 °C, 8″at 55 °C, 17″ at 72 °C for 45 cycles | 421 |
TNF-α | 5′-CAGAGGGAAGAGTTCCCCAG-3′ 5′-CCTTGGTCTGGTAGGAGACG-3′ | 5″ at 95 °C, 6″ at 57 °C, 13″ at 72 °C for 40 cycles | 324 |
TGF-β | 5′-CCGACTACTACGCCAAGGAGGTCAC-3′ 5′-AGGCCGGTTCATGCCATGAATGGTG-3′ | 5″ at 94 °C, 9″ at 60 °C, 18″ at 72 °C for 40 cycles | 439 |
HBD-2 | 5′-GGATCCATGGGTATAGGCGATCCTGTTA-3′ 5′-AAGCTTCTCTGATGAGGGAGCCCTTTCT-3′ | 5″ at 94 °C, 6″ at 63 °C, 10″ at 72 °C for 50 cycles | 198 |
Occludin | 5′-TCAGGGAATATCCACCTATCACTTCAG-3′ 5′-CATCAGCAGCAGCCATGTACTCTTCAC-3′ | 10″ at 95 °C, 45″ at 60 °C for 40 cycles | 188 |
Zonulin-1 | 5′-AGGGGCAGTGGTGGTTTTCTGTTCTTTC-3′ 5′-GCAGAGGTCAAAGTTCAAGGCTCAAGAGG-3′ | 10″ at 95 °C, 45″ at 60 °C for 40 cycles | 217 |
Claudin-1 | 5′-CTGGGAGGTGCCCTACTTTG-3′ 5′-ACACGTAGTCTTTCCCGCTG-3′ | 1″ at 95 °C, 30″ at 60 °C, 20″at 72 °C for 40 cycles | 128 |
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Fusco, A.; Savio, V.; Cimini, D.; D’Ambrosio, S.; Chiaromonte, A.; Schiraldi, C.; Donnarumma, G. In Vitro Evaluation of the Most Active Probiotic Strains Able to Improve the Intestinal Barrier Functions and to Prevent Inflammatory Diseases of the Gastrointestinal System. Biomedicines 2023, 11, 865. https://doi.org/10.3390/biomedicines11030865
Fusco A, Savio V, Cimini D, D’Ambrosio S, Chiaromonte A, Schiraldi C, Donnarumma G. In Vitro Evaluation of the Most Active Probiotic Strains Able to Improve the Intestinal Barrier Functions and to Prevent Inflammatory Diseases of the Gastrointestinal System. Biomedicines. 2023; 11(3):865. https://doi.org/10.3390/biomedicines11030865
Chicago/Turabian StyleFusco, Alessandra, Vittoria Savio, Donatella Cimini, Sergio D’Ambrosio, Adriana Chiaromonte, Chiara Schiraldi, and Giovanna Donnarumma. 2023. "In Vitro Evaluation of the Most Active Probiotic Strains Able to Improve the Intestinal Barrier Functions and to Prevent Inflammatory Diseases of the Gastrointestinal System" Biomedicines 11, no. 3: 865. https://doi.org/10.3390/biomedicines11030865