We investigated the the probiotic strains Lactobacillus rhamnosus, Lactobacillus reuteri, Bifidobacterium longum infantis and Lactobacillus helveticus for their ability to modulate cytokine release in primary murine mast cells in vitro

1. The pro– and anti–inflammatory
profiles of five different probiotics
Elizabeth Choi, Jacob Curtis, Laiba Jamshed, Rachael Wang
Honours Biology & Pharmacology, McMaster University

2. Background

3. The role of mast cells in IgE–mediated anaphylaxis
3
Mast Cells
● Granulocyte class of white blood cells1
● Originated from pluripotent progenitor cells of the bone marrow
● Found in mucosal and epithelial tissues
● Primary effector cells in allergic reactions
● Have surface FcεRI receptors
Immunoglobin E (IgE)
● Primary antibody involved in atopic and inflammatory diseases2
http://www.bloodjournal.org/content
/bloodjournal/112/4/946/F2.large.jpg

4. The mechanism of allergic reactions
4
Antigen
Granules
Crosslinking Degranulation
Figures adapted from Parham P (2014) The Immune System, 4th ed., Garland Science.

5. 5
Pro–inflammatory IL–6 vs. Anti–inflammatory IL–10
IL-6 IL-10
● Pro-inflammatory3
● Anti-inflammatory
● Necessary to stimulate
inflammation  fight infections
● Therapeutic target to decrease
inflammatory response
● Therapeutic target to increase
anti-inflammatory response
● Suppresses cytokine secretion 
attenuate inflammatory response

6. 6
The role of mast cells in host defense
● Have surface toll-like receptors (TLRs)4
● Gram-positive bacteria have peptidoglycan (PGN)  activates TLR-2
o Degranulation
● Gram-negative bacteria have lipopolysaccharide (LPS)  activates TLR-4
o No degranulation
Mast Cells
Both activated TLRs result in the release of IL-6 and IL-105

7. Adaptor
protein
Mast cell
co-receptor
TLR-4
7
The mechanism of TLR-4 activation
Figures adapted from Parham P (2014) The Immune System, 4th ed., Garland Science.
IL-6
Mast cell
IL-10
LPS
Gram-negative
bacterium

8. Discovery Project Overview

9. 9
The rationale of using probiotics
Probiotics
● Live microorganisms with beneficial health effects6
● Found naturally in the GI tract and externally in fermented foods
● Have been shown to reduce inflammatory cytokines in vitro7
o Effects are strain-specific
o May attenuate inflammation in allergic reactions and other
inflammatory diseases
Do probiotics have direct effects on mast cells?

10. 10
Objective
Hypothesis
To investigate the effects of different strains of probiotics on
cytokine release by bone marrow-derived mast cells (BMMCs) in vitro
Different strains of probiotics would differentially downregulate
IL–6 release and enhance IL–10 release

11. Experimental Procedures

12. 12
From PMMCs culturing to plating
4–6 weeks old
male BALB/c mice
BMMCs in pokeweed
mitogen-stimulated
culture medium
BMMCs splitting ×3
3 weeks later
BMMCs seedingELISAs
24 h
37 °C

13. 13
Enzyme-linked immunosorbent assay (ELISA)
A well in the 96-well microplate
Capture antibody
IL-6 or IL-10
Blocking agent
Detection antibody
Enzyme
Substrate
Figure adapted from https://www.thermofisher.com/content/dam/LifeTech/Images/integration/ELISAFormats575x214.jpg

14. 14
Advantages and limitations of ELISA
● Highly specific8
● Flexible
● Sensitive
● Have a specific antibody
● Present in detectable quantity
● Soluble in aqueous sample
● Cannot contain interfering
substances
Advantages Limitations

15. Results

16. Strains of probiotics tested
16
Gram-positive bacteria
● Lactobacillus rhamnosus (L. rhamnosus)
● Lactobacillus reuteri (L. reuteri)
● Bifidobacterium bifidum (B. bifidum)
● Bifidobacterium longum infantis (B. infantis)
● Lactobacillus helveticus (L. helveticus)

17. 17
Do probiotics attenuate IL–6 release?
L. rhamnosus L. reuteri

18. 18
Do probiotics attenuate IL–6 release?
B. bifidum
*
*
B. infantis
*
*
*
L. helveticus
* *

19. 19
Do L. rhamnosus and L. reuteri attenuate IL–6
release after LPS pre-treatment?

20. 20
Do probiotics enhance IL–10 release?
L. helveticusL. rhamnosus

21. 21
Do probiotics enhance IL–10 release?
B. infantis
L. reuteri
B. bifidum

22. Discussion

23. L. rhamnosus induced neither IL-6 nor IL-10 release
23
Figures adapted from Miettinen et al. (1998)
● IL-6 and IL-10 release from peripheral blood mononuclear cells (PBMCs)

24. L. reuteri induced the release of only IL-10
24
E. coli
L. reuteri
MF
Figure adapted from Smits et al. (2005) Figure adapted from Fink et al. (2007)
● IL-6 and IL-10 release from dendritic cells
*

25. Conclusion

26. Summary
26
Group IL-6 IL-10 Implication
Medium – – Negative control
– – InertL. rhamnosus
Similar to LPSB. infantis
– InflammatoryL. helveticus
Positive controlLPS
Anti-inflammatoryL. reuteri –
Similar to LPSB. bifidum

27. Conclusions and implications
27
● Different strains of probiotics have differential abilities to
modulate cytokine release
● L. reuteri appeared to be the most promising anti-inflammatory
agent
o In vivo experiments  potential supplementary treatment
● Probiotics have therapeutic potential in allergy and
inflammatory diseases

28. Acknowledgements
28
We would like to thank Dr. Paul Forsythe, from the Research
Institute of St. Joseph’s Hospital, for offering the opportunity to
complete this project in his lab and for his overall guidance
We would also like to thank Ibrahim Khambati and Catheryne Cao
for their constant assistance and support over the course of this
project

29. References
(1) Krystel-Whittemore M, Dileepan KN, and Wood JG (2015) Mast cell: a multi-functional master cell. Front Immunol 6:620.
(2) Stone KD, Prussin C, and Metcalfe DD (2010) IgE, mast cells, basophils, and eosinophils. J Allergy Clin Immunol 125:S73–S80.
(3) Zhang J-M, and An J (2007) Cytokines, inflammation, and pain. Int Anesthesiol Clin 45:27–37.
(4) Sandig H, and Bulfone-Paus S (2012) TLR signaling in mast cells: common and unique features. Front Immunol 3:185.
(5) Varadaradjalou S, Féger F, Thieblemont N, Hamouda NB, Pleau J-M, Dy M, and Arock M (2003) Toll-like receptor 2 (TLR2) and TLR4 differentially
activate human mast cells. Eur J Immunol 33:899–906.
(6) Shi LH, Balakrishnan K, Thiagarajah K, Mohd Ismail NI, and Yin OS (2016) Beneficial properties of probiotics. Trop life Sci Res 27:73–90.
(7) Michail S (2009) The role of probiotics in allergic diseases. Allergy Asthma Clin Immunol 5:5.
(8) The enzyme-linked immunosorbent assay (ELISA) (1976) . Bull World Health Organ 54:129–139.
(9) Miettinen M, Matikainen S, Vuopio-Varkila J, Pirhonen J, Varkila K, Kurimoto M, and Julkunen I (1998) Lactobacilli and streptococci induce
interleukin-12 (IL-12), IL-18, and gamma interferon production in human peripheral blood mononuclear cells. Infect Immun 66:6058–6062.
(10) Smits HH, Engering A, van der Kleij D, de Jong EC, Schipper K, van Capel TMM, Zaat BAJ, Yazdanbakhsh M, Wierenga EA, van Kooyk Y, and
Kapsenberg ML (2005) Selective probiotic bacteria induce IL-10–producing regulatory T cells in vitro by modulating dendritic cell function through
dendritic cell–specific intercellular adhesion molecule 3–grabbing nonintegrin. J Allergy Clin Immunol 115:1260–1267.
(11) Fink LN, Zeuthen LH, Christensen HR, Morandi B, Frokiaer H, and Ferlazzo G (2007) Distinct gut-derived lactic acid bacteria elicit divergent dendritic
cell-mediated NK cell responses. Int Immunol 19:1319–1327.
(12) Lammers KM, Brigidi P, Vitali B, Gionchetti P, Rizzello F, Caramelli E, Matteuzzi D, and Campieri M (2003) Immunomodulatory effects of probiotic
bacteria DNA: IL-1 and IL-10 response in human peripheral blood mononuclear cells. FEMS Immunol Med Microbiol 38:165–172. 30

30. Questions?

31. Supplementary Information

32. Does 1:100 PMMC : L. rhamnosus induce more
IL–6 release with increasing [LPS] pre-treatment?

33. B. Infantis induced both IL-6 and IL-10 release
33
Figures adapted from Lammers et al. (2003)
● IL-6 and IL-10 release from PBMCIL-6pg/PBMC
IL-10pg/PBMC