DEPARTMENT VIROLOGY, PARASITOLOGY AND IMMUNOLOGY RESEARCH GROUP IMMUNOLOGY THE EFFECTS OF β -1,3-1,6-GLUCANS ON INNATE IMMUNE RESPONSES IN PIGS . Hermans L., Devriendt B., Favoreel H., Cox E .
Haemolytic uremic syndrome STEC infection Renal failure Oedema Modulate Haemolytic anaemia Ataxia innate immune response in pigs Thrombocytopenia Death β -glucans Public health Economic losses C.L. Gyles (Ed.), Escherichia coli in Domestic Animals and Humans, CAB International, Wallingford, Oxon, UK (1994), p. 337 Wöchtl B et al. Comparison of clinical and immunological findings in gnotobiotic piglets infected with Escherichia coli O104 : H4 outbreak strain and EHEC O157 : H7. Gut Pathog. BioMed Central; 2017;1 – 18. Tseng M et al. Shiga toxin-producing Escherichia coli in swine: the public health perspective. Anim Health Res Rev. 2014;15(1):63 – 75.
β -glucans stimulate PBMC ➔ cytokine production ➔ NK cell activation
NK cells CD3 - Shape immune APC features CD172a - Cytotoxicity responses Memory-like PT CD8a + Rupert Handgretinger et al. Blood 2016;127:3341-3349 De Pelsmaeker et al. , J. Leukoc. Biol., 2018; 103(1); 129-140 Gerner et al., J Allergy Clin Immunol. 2013 September ; 132(3): 536 – 544
STEC INFECTION IN GNOTOBIOTIC PIGLETS 1. Control group 2. E. Coli O104:H4 infection 3. E. Coli O157:H7 infection Increase of NKp46 + NK cells in PBMC: IFN- γ production Potential role for NK cells during STEC infection Wöchtl B et al. Comparison of clinical and immunological findings in gnotobiotic piglets infected with Escherichia coli O104 : H4 outbreak strain and EHEC O157 : H7. Gut Pathog. BioMed Central; 2017;1 – 18
Beta-glucans?! PRR Proven to modulate ▪ Dectin-1 Carbohydrate polymers MAMP immunity ▪ CR3
FEED TRIAL WITH ETEC CHALLENGE Decrease in faecal ETEC excretion Lower antigen-specific IgM response In-feed administered β -glucans can protect against ETEC infection Stuyven et al., Vet Immunol Immunopathol, 2009; 128(1-3):60-6
Macrogard (Saccharomyces cerevisiae) STRUCTURE?! • Solubility Curdlan (Alcaligenes faecalis) • Size • Branching frequency • Length of the branches • Molecular conformation
Beta-glucans NK cells Activation Effects on innate immune responses
EFFECTS OF β– GLUCANS ON PORCINE NK CELLS
̶ ̶ ̶ ̶ Direct effects of β -glucans on pNK cells IL-2 NK isolation IL-2 Priming (16-18h) Beta-glucan (50 µg/ml) (2h) CFSE-labelled K562 (4h) Lysis of target cells
̶ ̶ ̶ ̶ Direct effects of β -glucans on pNK cells R e la tiv e N K -m e d ia te d ly s is NK isolation o f K 5 6 2 c e lls a fte r 1 8 h IL -2 p rim in g IL-2 Priming (16-18h) 1 .5 Beta-glucan (50 µg/ml) (2h) 1 .0 CFSE-labelled K562 (4h) 0 .5 0 .0 W ith o u t g lu c a n C u rd la n M a c ro g a rd No direct effect of β -glucans on pNK cells
̶ ̶ ̶ ̶ Indirect effects of β -glucans on pNK cells PBMC isolation β -glucan priming (10 µg/ml)/ M / IL-2 / IL-2 priming (50ng/ml) (18h) Medium (2h) CFSE-labelled K562 (4h) Lysis of target cells
̶ ̶ ̶ ̶ Indirect effects of β -glucans on pNK cells A b s o lu te P B M C -m e d ia te d ly s is * PBMC isolation * * * 8 0 β -glucan priming (10 µg/ml)/ IL-2 priming (50ng/ml) (18h) 6 0 4 0 Medium (2h) CFSE-labelled K562 (4h) 2 0 0 IL -2 M G C L M e d iu m MG: Macrogard β -glucans do indirectly activate pNK cells CL: Curdlan p<0,05: * ; p<0,001: ***
Are these effects NK-mediated? 1 5 R e la tiv e ly s is 1 0 5 0 P B M C P B M C N K P B M C P B M C N K P B M C P B M C N K P B M C P B M C N K M e d iu m IL -2 M G C L (1 0 µ g /m l) (1 0 µ g /m l) Yes
HOW DOES THIS WORK?
̶ ̶ ̶ ̶ ̶ Effect of supernatant on pNK cells PBMC isolation M / IL-2 / β -glucan priming (10 µg/ml)/ IL-2 priming (50ng/ml) (18h) Collecting culture SN FACS purified NK cells (18h) CFSE-labelled K562 cells (4h) SN
̶ ̶ ̶ ̶ ̶ Effect of supernatant on pNK cells A b s o lu te N K -m e d ia te d ly s is PBMC isolation * β -glucan priming (10 µg/ml)/ * * * 1 0 0 IL-2 priming (50ng/ml) (18h) 8 0 6 0 4 0 Collecting culture SN 2 0 FACS purified NK cells (18h) CFSE-labelled K562 cells (4h) 0 2 G L M - C M L I p<0,05: * ; p<0,001: *** PBMC produce NK cell stimulating factors upon β -glucan priming
Effect of monocytes/DC P B M C -m e d ia te d ly s is o f K 5 6 2 c e lls *** 5 0 4 0 % ly s e 3 0 ** 2 0 1 0 0 + + + l l l l l l a a a u u u 2 2 2 F F F 7 7 7 1 1 1 D D D C C C M e d ium M G IL -2 (1 0 µ g /m l) Role for CD172a+ cells: monocytes and/or DC p<0,01: ** ; p<0,001: ***
CONCLUSIONS
Monocytes / DC Beta-glucans Cytokines Cytotoxicity Cytokines ??? Maturation of DC Activation of monocytes Activation of Th1
Monocytes / DC Beta-glucans Cytokines Cytotoxicity Cytokines ??? Shape innate immune response → Stronger resistance to infection Maturation of DC Activation of monocytes Activation of Th1
Leen Hermans DEPARTMENT VIROLOGY, PARASITOLOGY AND IMMUNOLOGY Acknowledgements Promotors: Prof. Eric Cox, Prof. Herman Favoreel Co-promotor: Dr. Bert Devriendt ICONS AND FIGURES Post Pigeon by Alina Oleynik from the Noun Project Mail Password by iconsmind.com from the Noun Project Human by Rose Alice Design from the Noun Project BioRender
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