Advertisement

In Vivo Implications of Potential Probiotic Lactobacillus reuteri LR6 on the Gut and Immunological Parameters as an Adjuvant Against Protein Energy Malnutrition

  • Sheenam GargEmail author
  • Tejinder P. Singh
  • Ravinder K. Malik
Article

Abstract

The present study investigated the impact of probiotic Lactobacillus reuteri LR6 on the gut and systemic immunity using protein energy malnourished (PEM) murine model. Thirty male Swiss albino mice were divided into five groups: control (C), malnourished (M), probiotic fermented milk (PFM), skim milk (SM), and bacterial suspension (BS) with six mice per group. Group C was fed with conventional diet throughout the study while the other groups were fed with protein calorie restricted diet until the development of malnutrition. After development of malnutrition, group M was continued with the restricted diet while other groups were fed with re-nourished diet supplemented with PFM, SM, and BS for 1 week, respectively. Thereafter, mice were sacrificed and different histological, microbiological, and immunological parameters were studied. Probiotics feeding in PEM model as fermented product or bacterial suspension improved the intestinal health in terms of intact morphology of colonic crypts, normal goblet cells, and intact lamina propria with no inflammation in large intestine, absence of fibrosis, and no inflammation in spleen. The number of secretory IgA+ cells was significantly higher in group PFM and BS. Also, increase in the phagocytic percentage of the macrophages and bone marrow derived dendritic cells (DCs) were observed in the PFM and BS group in comparison to the group M. In comparison to the group M and SM, lactobacilli, bifidobacteria, and Firmicutes counts were significantly higher in the group PFM and BS. This study concludes that probiotic supplementation to re-nutrition diet could emerge as wonder therapeutics against PEM.

Keywords

Probiotics Protein energy malnutrition Lactobacillus reuteri Gut microbiota changes Immunocytochemistry 

Notes

Acknowledgments

We thank Dr. Suman Kapila, Dr. Sachinandan De, and Dr. S. K. Tomar for the technical assistance in their respective laboratories.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that there is no conflict of interest.

References

  1. 1.
    Garg S, Malik RK, Singh TP, Renuka (2014) Child nutrition: a pillar to development. IJAR 2(1):766–772Google Scholar
  2. 2.
    Ghosh TS, Gupta SS, Bhattacharya T et al (2014) Gut microbiomes of Indian children of varying nutritional status. PLoS One 9(4):e95547Google Scholar
  3. 3.
    Gupta SS, Mohammed MH, Ghosh TS, Kanungo S, Nair GB, Mande SS (2011) Metagenome of the gut of a malnourished child. Gut Pathog 3(1):7Google Scholar
  4. 4.
    Kane AV, Dinh DM, Ward HD (2014) Childhood malnutrition and the intestinal microbiome. Pediatr Res 77:256–262Google Scholar
  5. 5.
    Kau AL, Ahern PP, Griffin NW, Goodman AL, Gordon JI (2011) Human nutrition, the gut microbiome and the immune system. Nature 474(7351):327–336Google Scholar
  6. 6.
    Lalles JP (2012) Long term effects of pre-and early postnatal nutrition and environment on the gut. J Anim Sci 90(4):421–429Google Scholar
  7. 7.
    Million M, Diallo A, Raoult D (2017) Gut microbiota and malnutrition. Microb Pathog 106:127–138Google Scholar
  8. 8.
    Monira S, Nakamura S, Gotoh K et al (2011) Gut microbiota of healthy and malnourished children in Bangladesh. Front Microbiol 2:228Google Scholar
  9. 9.
    Preidis GA, Ajami NJ, Wong MC, Bessard BC, Conner ME, Petrosino JF (2015) Composition and function of the undernourished neonatal mouse intestinal microbiome. J Nutr Biochem 26(10):1050–1057Google Scholar
  10. 10.
    Smith MI, Yatsunenko T, Manary MJ, Trehan I, Mkakosya R, Cheng J, Kau AL, Rich SS, Concannon P, Mychaleckyj JC, Liu J, Houpt E, Li JV, Holmes E, Nicholson J, Knights D, Ursell LK, Knight R, Gordon JI (2013) Gut microbiomes of Malawian twin pairs discordant for kwashiorkor. Science 339(6119):548–554Google Scholar
  11. 11.
    Tilg H, Moschen AR (2013) Gut microbiota: malnutrition and microbiota—a new relationship? Nat Rev Gastroenterol Hepatol 10(5):261–262Google Scholar
  12. 12.
    de Azevedo JF, Hermes C, Manzano MA et al (2007) Análise morfométrica da parede intestinal do íleo de ratos submetidos a intensa carência de proteínas. Arq Ciênc Vet Zool Unipar 10(2):85–90Google Scholar
  13. 13.
    França TG, Ishikawa LL, Zorzella-Pezavento SF, Chiuso-Minicucci F, da Cunha MLRS, Sartori A (2009) Impact of malnutrition on immunity and infection. J Venom Anim Toxins Incl Trop Dis 15(3):374–390Google Scholar
  14. 14.
    Galdeano CM, Núñez IN, de LeBlanc AD et al (2011) Impact of a probiotic fermented milk in the gut ecosystem and in the systemic immunity using a non-severe protein-energy-malnutrition model in mice. BMC Gastroenterol 11(1):64Google Scholar
  15. 15.
    Gurmini J, Cecílio WA, Schuler SL et al (2005) In-uterus malnutrition and its changes in the small bowel of Wistar rats at birth and after lactation. J Bras Patol Med Lab 41(4):271–278Google Scholar
  16. 16.
    Hermes C, Azevedo JF, Araújo EJ et al (2008) Intestinal ascending colon morphometrics in rats submitted to severe protein malnutrition. Int J Morphol 26(1):5–11Google Scholar
  17. 17.
    de Melo JF, Da Costa TB, da Costa Lima TD et al (2013) Long-term effects of a neonatal low-protein diet in rats on the number of macrophages in culture and the expression/production of fusion proteins. Eur J Nutr 52(5):1475–1482Google Scholar
  18. 18.
    Gonzalez B, Guerra C, Morris D et al (2010) Dendritic cells in infectious disease, hypersensitivity, and autoimmunity. Int J Interferon Cytokine Mediat Res 2(1):137–147Google Scholar
  19. 19.
    Hughes SM, Amadi B, Mwiya M, Nkamba H, Tomkins A, Goldblatt D (2009) Dendritic cell energy results from endotoxemia in severe malnutrition. J Immunol 183(4):2818–2826Google Scholar
  20. 20.
    Ibrahim MK, Barnes JL, Osorio EY, Anstead GM, Jimenez F, Osterholzer JJ, Travi BL, Ahuja SS, White AC Jr, Melby PC (2014) Deficiency of lymph node-resident dendritic cells (DCs) and dysregulation of DC chemoattractants in a malnourished mouse model of Leishmania donovani infection. Infect Immun 82(8):3098–3112Google Scholar
  21. 21.
    Mello AS, de Oliveira DC, Bizzarro B, Sá-Nunes A, Hastreiter AA, de Oliveira Beltran JS, Xavier JG, Borelli P, Fock RA (2014) Protein malnutrition alters spleen cell proliferation and IL-2 and IL-10 production by affecting the STAT-1 and STAT-3 balance. Inflammation 37(6):2125–2138Google Scholar
  22. 22.
    Stapleton PP, Fujita J, Murphy EM, Naama HA, Daly JM (2001) The influence of restricted calorie intake on peritoneal macrophage function. Nutrition 17(1):41–45Google Scholar
  23. 23.
    Suskind RM, Tontisirin K, Nestlé S (2001) Nutrition, immunity, and infection in infants and children. Lippincott Williams & WilkinsGoogle Scholar
  24. 24.
    Allori C, Agüero G, de Ruiz Holgado AP et al (2000) Gut mucosa morphology and microflora changes in malnourished mice after renutrition with milk and administration of Lactobacillus casei. J Food Prot 63(1):83–90Google Scholar
  25. 25.
    Cano PG, Aguero G, Perdigon GA (2002) Adjuvant effects of Lactobacillus casei added to a renutrition diet in a malnourished mouse model. Biocell 26(1):35–48Google Scholar
  26. 26.
    Christensen HR, Frøkiær H, Pestka JJ (2002) Lactobacilli differentially modulate expression of cytokines and maturation surface markers in murine dendritic cells. J Immunol 168(1):171–178Google Scholar
  27. 27.
    de LeBlanc AD, Chaves S, Carmuega E et al (2008) Effect of long-term continuous consumption of fermented milk containing probiotic bacteria on mucosal immunity and the activity of peritoneal macrophages. Immunobiology 213(2):97–108Google Scholar
  28. 28.
    Foligne B, Zoumpopoulou G, Dewulf J, Ben Younes A, Chareyre F, Sirard JC, Pot B, Grangette C (2007) A key role of dendritic cells in probiotic functionality. PLoS One 2(3):e313Google Scholar
  29. 29.
    Hart AL, Lammers K, Brigidi P, Vitali B, Rizzello F, Gionchetti P, Campieri M, Kamm MA, Knight SC, Stagg AJ (2004) Modulation of human dendritic cell phenotype and function by probiotic bacteria. Gut 53(11):1602–1609Google Scholar
  30. 30.
    Jain S, Yadav H, Sinha PR (2008) Stimulation of innate immunity by oral administration of dahi containing probiotic Lactobacillus casei in mice. J Med Food 11(4):652–656Google Scholar
  31. 31.
    Kapila R, Kapila S, Kapasiya M, Pandey D, Dang A, Saliganti V (2012) Comparative evaluation of oral administration of probiotic lactobacilli-fermented milks on macrophage function. Probiotics Antimicrob Proteins 4(3):173–179Google Scholar
  32. 32.
    Kapila R, Sebastian R, Varma D et al (2013) Comparison of innate immune activation after prolonged feeding of milk fermented with three species of lactobacilli. Microbiol Immunol 57(11):778–784Google Scholar
  33. 33.
    Mohamadzadeh M, Olson S, Kalina WV, Ruthel G, Demmin GL, Warfield KL, Bavari S, Klaenhammer TR (2005) Lactobacilli activate human dendritic cells that skew T cells toward T helper 1 polarization. Proc Natl Acad Sci U S A 102(8):2880–2885Google Scholar
  34. 34.
    Paturi G, Phillips M, Kailasapathy K (2008) Effect of probiotic strains Lactobacillus acidophilus LAFTI L10 and Lactobacillus paracasei LAFTI L26 on systemic immune functions and bacterial translocation in mice. J Food Prot 71(4):796–801Google Scholar
  35. 35.
    Singh TP, Kaur G, Malik RK, Schillinger U, Guigas C, Kapila S (2012) Characterization of intestinal Lactobacillus reuteri strains as potential probiotics. Probiotics Antimicrob Proteins 4(1):47–58Google Scholar
  36. 36.
    Singh TP, Malik RK, Kaur G (2016) Cell surface proteins play an important role in probiotic activities of Lactobacillus reuteri. Nutrire 41(1):5Google Scholar
  37. 37.
    Singh TP, Kaur G, Kapila S et al (2017) Antagonistic activity of lactobacillus reuteri strains on the adhesion characteristics of selected pathogens. Front Microbiol 8:486Google Scholar
  38. 38.
    Singh TP, Malik RK, Katkamwar SG, Kaur G (2015) Hypocholesterolemic effects of Lactobacillus reuteri LR6 in rats fed on high-cholesterol diet. Int J Food Sci Nutr 66(1):71–75Google Scholar
  39. 39.
    Garg S, Singh TP, Reddi S, Malik RK, Kapila S (2017) Intervention of probiotic Lb. reuteri fermented milk as an adjuvant to combat protein energy malnourishment induced gut disturbances in albino mice. J Funct Foods 36:467–479Google Scholar
  40. 40.
    AOAC. Official method of analysis. Association of official agric chemists (1984) p. AOAC, Washington, DC, p 988Google Scholar
  41. 41.
    Kiernan JA (2008) Histological and histochemical methods theory and practice. (4th edn.), Scion, BloxhamGoogle Scholar
  42. 42.
    Inaba K, Swiggard WJ, Steinman RM et al (2009) Isolation of dendritic cells. Curr Protoc Immunol 19:3–7Google Scholar
  43. 43.
    Hashizume T, Imayama S, Hori Y (1992) Scanning electron microscopic study on dendritic cells and fibroblasts in connective tissue. Microscopy 41(6):434–437Google Scholar
  44. 44.
    Rinttilä T, Kassinen A, Malinen E et al (2004) Development of an extensive set of 16S rDNA-targeted primers for quantification of pathogenic and indigenous bacteria in faecal samples by real-time PCR. J Appl Microbiol 97(6):1166–1177Google Scholar
  45. 45.
    Hu X, Wang T, Li W, Jin F, Wang L (2013) Effects of NS Lactobacillus strains on lipid metabolism of rats fed a high-cholesterol diet. Lipids Health Dis 12(1):67Google Scholar
  46. 46.
    Abdulamir AS, Yoke TS, Nordin N et al (2010) Detection and quantification of probiotic bacteria using optimized DNA extraction, traditional and real-time PCR methods in complex microbial communities. AJB 9(10):1481–1492Google Scholar
  47. 47.
    De Azevedo JF, Hermes-Uliana C, Lima DP et al (2014) Probiotics protect the intestinal wall of morphological changes caused by malnutrition. An Acad Bras Ciênc 86(3):1303–1314Google Scholar
  48. 48.
    Dock DB, Aguilar-Nascimento JE, Latorraca MQ (2003) Enhanced immunological response influenced by probiotics during the recovery of experimental malnutrition. Revista Brasileira de Nutrição Clínica 18:157–162Google Scholar
  49. 49.
    Dock DB, Aguilar-Nascimento JE, Latorraca MQ (2004) Probiotics enhance the recovery of gut atrophy in experimental malnutrition. Biocell 28(2):143–150Google Scholar
  50. 50.
    Lima DP, Azevedo JFD, Hermes-Uliana C et al (2012) Probiotics prevent growth deficit of colon wall strata of malnourished rats post-lactation. An Acad Bras Ciênc 84(3):727–736Google Scholar
  51. 51.
    Olusi SO, McFarlane H (1976) Effects of early protein-calorie malnutrition on the immune response. Pediatr Res 10(8):707–712Google Scholar
  52. 52.
    Faulk WP, Paes RP, Marigo C (1976) The immunological system in health and malnutrition. Proc Nutr Soc 35(3):253–261Google Scholar
  53. 53.
    Chandra RK (1997) Nutrition and the immune system: an introduction. Am J Clin Nutr 66(2):460S–463SGoogle Scholar
  54. 54.
    Kemgang TS, Kapila S, Shanmugam VP, Kapila R (2014) Cross-talk between probiotic lactobacilli and host immune system. J Appl Microbiol 117(2):303–319Google Scholar
  55. 55.
    Berman SH, Eichelsdoerfer P, Yim D, Elmer GW, Wenner CA (2006) Daily ingestion of a nutritional probiotic supplement enhances innate immune function in healthy adults. Nutr Res 26(9):454–459Google Scholar
  56. 56.
    Galdeano CM, Perdigon G (2006) The probiotic bacterium Lactobacillus casei induces activation of the gut mucosal immune system through innate immunity. Clin Vaccine Immunol 13(2):219–226Google Scholar
  57. 57.
    Ha CL, Woodward B (1997) Reduction in the quantity of the polymeric immunoglobulin receptor is sufficient to account for the low concentration of intestinal secretory immunoglobulin a in a weanling mouse model of wasting protein-energy malnutrition. J Nutr 127(3):427–435Google Scholar
  58. 58.
    Mizumachi K, Aoki R, Ohmori H, Saeki M, Kawashima T (2009) Effect of fermented liquid diet prepared with Lactobacillus plantarum LQ80 on the immune response in weaning pigs. Animal 3(5):670–676Google Scholar
  59. 59.
    Ohland CL, MacNaughton WK (2010) Probiotic bacteria and intestinal epithelial barrier function. Am J Physiol Gastrointest Liver Physiol 298(6):G807–G819Google Scholar
  60. 60.
    Paturi G, Phillips M, Jones M et al (2007) Immune enhancing effects of Lactobacillus acidophilus LAFTI L10 and Lactobacillus paracasei LAFTI L26 in mice. Int J Food Microbiol 115(1):115–118Google Scholar
  61. 61.
    Rytter MJ, Kolte L, Briend A et al (2014) The immune system in children with malnutrition—a systematic review. PLoS One 9(8):e105017Google Scholar
  62. 62.
    Watson RR, McMurray DN, Martin P et al (1985) Effect of age, malnutrition and renutrition on free secretory component and IgA in secretions. Am J Clin Nutr 42(2):281–288Google Scholar
  63. 63.
    Welsh FK, Farmery SM, MacLennan K et al (1998) Gut barrier function in malnourished patients. Gut 42(3):396–401Google Scholar
  64. 64.
    Afacan NJ, Fjell CD, Hancock RE (2012) A systems biology approach to nutritional immunology–focus on innate immunity. Mol Asp Med 33(1):14–25Google Scholar
  65. 65.
    Marranzino G, Villena J, Salva S, Alvarez S (2012) Stimulation of macrophages by immunobiotic Lactobacillus strains: influence beyond the intestinal tract. Microbiol Immunol 56(11):771–781Google Scholar
  66. 66.
    Steinman RM, Hawiger D, Nussenzweig MC (2003) Tolerogenic dendritic cells. Annu Rev Immunol 21(1):685–711Google Scholar
  67. 67.
    Abe M, Akbar F, Matsuura B, Horiike N, Onji M (2003) Defective antigen-presenting capacity of murine dendritic cells during starvation. Nutrition 19(3):265–269Google Scholar
  68. 68.
    Tsai YT, Cheng PC, Fan CK, Pan TM (2008) Time-dependent persistence of enhanced immune response by a potential probiotic strain Lactobacillus paracasei subsp. paracasei NTU 101. Int J Food Microbiol 128(2):219–225Google Scholar
  69. 69.
    Mueller DL, Jenkins MK, Schwartz RH (1989) Clonal expansion versus functional clonal inactivation: a costimulatory signalling pathway determines the outcome of T cell antigen receptor occupancy. Annu Rev Immunol 7(1):445–480Google Scholar
  70. 70.
    Damoiseaux JG, Yagita H, Okumura K et al (1998) Costimulatory molecules CD80 and CD86 in the rat; tissue distribution and expression by antigen-presenting cells. J Leukoc Biol 64(6):803–809Google Scholar
  71. 71.
    Cai S, Kandasamy M, Rahmat JN et al (2016) Lactobacillus rhamnosus GG activation of dendritic cells and neutrophils depends on the dose and time of exposure. J Immunol Res 2016:740–760Google Scholar
  72. 72.
    Xing F, Wang J, Hu M, Yu Y, Chen G, Liu J (2011) Comparison of immature and mature bone marrow-derived dendritic cells by atomic force microscopy. Nanoscale Res Lett 6(1):455Google Scholar
  73. 73.
    Dock-Nascimento DB, Junqueira K, Aguilar-Nascimento JE (2007) Rapid restoration of colonic goblet cells induced by a hydrolyzed diet containing probiotics in experimental malnutrition. Acta Cir Bras 22:72–76Google Scholar
  74. 74.
    de LeBlanc ADM, LeBlanc JG (2014) Effect of probiotic administration on the intestinal microbiota, current knowledge and potential applications. World J Gastroenterol 20(33):16518–16528Google Scholar
  75. 75.
    Gareau MG, Sherman PM, Walker WA (2010) Probiotics and the gut microbiota in intestinal health and disease. Nat Rev Gastroenterol Hepatol 7(9):503–514Google Scholar
  76. 76.
    Hemarajata P, Versalovic J (2013) Effects of probiotics on gut microbiota: mechanisms of intestinal immunomodulation and neuromodulation. Ther Adv Gastroenterol 6(1):39–51Google Scholar
  77. 77.
    Kootte RS, Vrieze A, Holleman F, Dallinga-Thie GM, Zoetendal EG, de Vos WM, Groen AK, Hoekstra JBL, Stroes ES, Nieuwdorp M (2012) The therapeutic potential of manipulating gut microbiota in obesity and type 2 diabetes mellitus. Diabetes Obes Metab 14(2):112–120Google Scholar
  78. 78.
    Sanz Y (2011) Gut microbiota and probiotics in maternal and infant health. The Am J Clin Nutr 94(6):2000S–2005SGoogle Scholar
  79. 79.
    Scaldaferri F, Gerardi V, Lopetuso LR, del Zompo F, Mangiola F, Boškoski I, Bruno G, Petito V, Laterza L, Cammarota G, Gaetani E, Sgambato A, Gasbarrini A (2013) Gut microbial flora, prebiotics, and probiotics in IBD: their current usage and utility. Biomed Res Int 2013 (Article ID 435268:1–9Google Scholar
  80. 80.
    Walsh CJ, Guinane CM, O’Toole PW et al (2014) Beneficial modulation of the gut microbiota. FEBS Letts 588(22):4120–4130Google Scholar
  81. 81.
    Wang S, Zhu H, Lu C, Kang Z, Luo Y, Feng L, Lu X (2012) Fermented milk supplemented with probiotics and prebiotics can effectively alter the intestinal microbiota and immunity of host animals. J Dairy Sci 95(9):4813–4822Google Scholar
  82. 82.
    Zakostelska Z, Kverka M, Klimesova K, Rossmann P, Mrazek J, Kopecny J, Hornova M, Srutkova D, Hudcovic T, Ridl J, Tlaskalova-Hogenova H (2011) Lysate of probiotic Lactobacillus casei DN-114 001 ameliorates colitis by strengthening the gut barrier function and changing the gut microenvironment. PLoS One 6(11):e27961Google Scholar
  83. 83.
    Humen MA, De Antoni GL, Benyacoub J et al (2005) Lactobacillus johnsonii La1 antagonizes Giardia intestinalis in vivo. Infect Immun 73(2):1265–1269Google Scholar
  84. 84.
    Ren ZG, Liu H, Jiang JW, Jiang L, Chen H, Xie HY, Zhou L, Zheng SS (2011) Protective effect of probiotics on intestinal barrier function in malnourished rats after liver transplantation. HBPD Int 10(5):489–496Google Scholar
  85. 85.
    Shukla G, Devi P, Sehgal R (2008) Effect of Lactobacillus casei as a probiotic on modulation of giardiasis. Dig Dis Sci 53(10):2671–2679Google Scholar
  86. 86.
    Shukla G, Kaur T, Sehgal R et al (2010) Protective potential of L. acidophilus in murine giardiasis. Open Med 5(4):456–463Google Scholar
  87. 87.
    Shukla G, Sidhu RK (2011) Lactobacillus casei as a probiotic in malnourished Giardia lamblia-infected mice: a biochemical and histopathological study. Can J Microbiol 57(2):127–135Google Scholar
  88. 88.
    Villena J, Racedo S, Agüero G, Alvarez S (2006) Yoghurt accelerates the recovery of defence mechanisms against Streptococcus pneumoniae in protein-malnourished mice. Br J Nutr 95(3):591–602Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Sheenam Garg
    • 1
    Email author
  • Tejinder P. Singh
    • 2
  • Ravinder K. Malik
    • 1
  1. 1.Dairy Microbiology DivisionNational Dairy Research InstituteKarnalIndia
  2. 2.Dairy Microbiology Department, College of Dairy Science and TechnologyLala Lajpat Rai University of Veterinary and Animal ScienceHisarIndia

Personalised recommendations